WorldWideScience

Sample records for skin fibroblast lines

  1. Derivation of Human Skin Fibroblast Lines for Feeder Cells of Human Embryonic Stem Cells.

    Science.gov (United States)

    Unger, Christian; Felldin, Ulrika; Rodin, Sergey; Nordenskjöld, Agneta; Dilber, Sirac; Hovatta, Outi

    2016-02-03

    After the first derivations of human embryonic stem cell (hESC) lines on fetal mouse feeder cell layers, the idea of using human cells instead of mouse cells as feeder cells soon arose. Mouse cells bear a risk of microbial contamination, and nonhuman immunogenic proteins are absorbed from the feeders to hESCs. Human skin fibroblasts can be effectively used as feeder cells for hESCs. The same primary cell line, which can be safely used for up to 15 passages after stock preparations, can be expanded and used for large numbers of hESC derivations and cultures. These cells are relatively easy to handle and maintain. No animal facilities or animal work is needed. Here, we describe the derivation, culture, and cryopreservation procedures for research-grade human skin fibroblast lines. We also describe how to make feeder layers for hESCs using these fibroblasts.

  2. Outgrowth of fibroblast cells from goat skin explants in three different culture media and the establishment of cell lines.

    Science.gov (United States)

    Singh, Mahipal; Sharma, Anil K

    2011-02-01

    Three different commercially available media, known to support human and porcine-specific fibroblast cultures, were tested for their growth potential on goat skin explants. Although outgrowth of fibroblasts was observed in all media tested, irrespective of breed, porcine-specific media exhibited higher rate of growth. Using this media, three fibroblast cell lines (GSF289, GSF737, and GSF2010) from ear skin explants of normal healthy dairy goats of Kiko and Saanen breed were successfully established in culture. Liquid nitrogen stocks of these frozen cells had a viability rate of 96.2% in in vitro cultures. These cells were morphologically indistinguishable from the cell stocks prior to freezing. Analysis of the growth of a fifth passage culture revealed an 'S' shaped growth curve with a population doubling time of 25 h. The cell lines were found negative for microbial, fungal, and mycoplasma contaminations. These goat skin fibroblast lines and the simple method of their isolation and freezing with high rate of viability will provide additional tools to study molecular mechanisms that regulate fibroblast function and for genetic manipulation of small ruminants.

  3. [The influence of substrate from extracellular matrix proteins on karyotypic variability of the Indian muntjac skin fibroblast two cell lines].

    Science.gov (United States)

    Polianskaia, G G; Kol'tsova, A M

    2013-01-01

    The effect of cell culture conditions on numerical and structural karyotypic variability was investigated in two Indian muntjac skin fibroblast "markerless" cell lines, M and MT. The cells cultivated on the substrate consisting of extracellular matrix proteins (ECM), synthesized by human mesenchymal stem cells (SC5-MSC). The character of cell distribution for chromosome number of cell line M changed after cultivation for 1 and 4 days as compared to control cells, which were cultured on hydrophilic surface without ECM-coating. These changes involve a significant decrease in frequency of cells with modal numbers of chromosomes and an increase in frequency of cells with lower chromosome numbers. Many new types of additional structural variants of the karyotype (SVK) appear. MT cell line, differing from M line in the number of homologous chromosomes, demonstrated similar with M line the character of cell distribution for chromosome number only for 1 day after cultivating on the ECM-substrate, but not after 4 days in the same culture conditions, no difference from the control cells was observed. The observed alterations seem to be due to disturbances in correct chromosome segregation process, which were caused by abrupt shift in the cell culture conditions. The analysis of the structural karyotypic variability revealed significant increase in frequency of chromosomal aberrations in M cell line for 1 and 4 days in culture on the ECM-substrate as compared to the control cells. The frequency of dicentric chromosomes (telomeric associations) was increased and constituted more than 50% of all chromosome aberrations. No increase in frequency of chromosome aberrations was observed for MT cells cultured in the same conditions. The obtained results show that the cell lines of the same origin but of different karyotypic structure react to substrate in a different way. In contrast to M line, in MT line a fast normalization of numerical karyotypic characteristics and no enhancement

  4. Alteration of Skin Properties with Autologous Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Rajesh L. Thangapazham

    2014-05-01

    Full Text Available Dermal fibroblasts are mesenchymal cells found between the skin epidermis and subcutaneous tissue. They are primarily responsible for synthesizing collagen and glycosaminoglycans; components of extracellular matrix supporting the structural integrity of the skin. Dermal fibroblasts play a pivotal role in cutaneous wound healing and skin repair. Preclinical studies suggest wider applications of dermal fibroblasts ranging from skin based indications to non-skin tissue regeneration in tendon repair. One clinical application for autologous dermal fibroblasts has been approved by the Food and Drug Administration (FDA while others are in preclinical development or various stages of regulatory approval. In this context, we outline the role of fibroblasts in wound healing and discuss recent advances and the current development pipeline for cellular therapies using autologous dermal fibroblasts. The microanatomic and phenotypic differences of fibroblasts occupying particular locations within the skin are reviewed, emphasizing the therapeutic relevance of attributes exhibited by subpopulations of fibroblasts. Special focus is provided to fibroblast characteristics that define regional differences in skin, including the thick and hairless skin of the palms and soles as compared to hair-bearing skin. This regional specificity and functional identity of fibroblasts provides another platform for developing regional skin applications such as the induction of hair follicles in bald scalp or alteration of the phenotype of stump skin in amputees to better support their prosthetic devices.

  5. Differences in motility pattern between human buccal fibroblasts and periodontal and skin fibroblasts

    DEFF Research Database (Denmark)

    Lepekhin, Eugene; Grøn, Birgitte; Berezin, Vladimir

    2002-01-01

    Migration of fibroblasts from surrounding normal tissue into the wound bed is an important requirement for successful wound healing. This study investigated the motility pattern of buccal, periodontal and skin fibroblasts to determine whether differences in the wound healing efficiency...... at these sites can be explained by differences in the motile behavior of their respective fibroblast populations. The migratory characteristics were studied in a two-dimensional culture system. The migration of single cells was time-lapse video recorded at intervals of 15 min for a period of 6 h using a computer...... displacement of periodontal and skin fibroblasts. The decreased cellular displacement of the buccal fibroblasts was found to be due to both lower cellular speed and less persistence in direction. The buccal fibroblasts also displayed smaller areas and longer processes. The differences in cellular morphology...

  6. Respiratory activity and growth of human skin derma fibroblasts.

    Science.gov (United States)

    Papa, F; Scacco, S; Vergari, R; Bucaria, V; Dioguardi, D; Papa, S

    1998-09-01

    A study has been made on the speed of growth and respiratory activity of fibroblast cultures from control derma, cheloid (hypertrophic) scar and stabilized scar taken from human skin. The speed of growth and the efficiency of plaque formation of fibroblasts from cheloid scar were greater in comparison with those of fibroblasts from stabilized scar and were stimulated by the addition to the culture medium of the exudate from post-traumatic ulcer. Measurement of the contents of cytochromes showed a decrease in the content of cytochromes b562 and c + c1 in the fibroblast culture from both cheloid and stabilized scar as compared to the fibroblast culture from control derma. Cytochrome aa3 content did not show significant difference among the three types of fibroblast cultures. The respiratory activities supported by pyruvate plus malate, succinate or ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine did not show, however, significant difference among the three fibroblast cultures. These observations show that the speed of growth of skin fibroblasts does not depend on the overall respiratory capacity. The exudate stimulated the activity of cytochrome c oxidase in fibroblasts from control derma, and cheloid scar. This effect and the accompanying stimulation of fibroblast growth might be correlated with the balance of oxygen free radicals.

  7. Ginseng-berry-mediated gold and silver nanoparticle synthesis and evaluation of their in vitro antioxidant, antimicrobial, and cytotoxicity effects on human dermal fibroblast and murine melanoma skin cell lines

    Science.gov (United States)

    Jiménez Pérez, Zuly Elizabeth; Mathiyalagan, Ramya; Markus, Josua; Kim, Yeon-Ju; Kang, Hyun Mi; Abbai, Ragavendran; Seo, Kwang Hoon; Wang, Dandan; Soshnikova, Veronika; Yang, Deok Chun

    2017-01-01

    There has been a growing interest in the design of environmentally affable and biocompatible nanoparticles among scientists to find novel and safe biomaterials. Panax ginseng Meyer berries have unique phytochemical profile and exhibit beneficial pharmacological activities such as antihyperglycemic, antiobesity, antiaging, and antioxidant properties. A comprehensive study of the biologically active compounds in ginseng berry extract (GBE) and the ability of ginseng berry (GB) as novel material for the biosynthesis of gold nanoparticles (GBAuNPs) and silver nanoparticles (GBAgNPs) was conducted. In addition, the effects of GBAuNPs and GBAgNPs on skin cell lines for further potential biological applications are highlighted. GBAuNPs and GBAgNPs were synthesized using aqueous GBE as a reducing and capping agent. The synthesized nanoparticles were characterized for their size, morphology, and crystallinity. The nanoparticles were evaluated for antioxidant, anti-tyrosinase, antibacterial, and cytotoxicity activities and for morphological changes in human dermal fibroblast and murine melanoma skin cell lines. The phytochemicals contained in GBE effectively reduced and capped gold and silver ions to form GBAuNPs and GBAgNPs. The optimal synthesis conditions (ie, temperature and v/v % of GBE) and kinetics were investigated. Polysaccharides and phenolic compounds present in GBE were suggested to be responsible for stabilization and functionalization of nanoparticles. GBAuNPs and GBAgNPs showed increased scavenging activity against 2,2-diphenyl-1-picrylhydrazyl free radicals compared to GBE. GBAuNPs and GBAgNPs effectively inhibited mushroom tyrosinase, while GBAgNPs showed antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, GBAuNPs were nontoxic to human dermal fibroblast and murine melanoma cell lines, and GBAgNPs showed cytotoxic effect on murine melanoma cell lines. The current results evidently suggest that GBAgNPs can act as potential

  8. Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts

    NARCIS (Netherlands)

    C.M.A. Reijnders; A. van Lier; S. Roffel; D. Kramer; R.J. Scheper; S. Gibbs

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  9. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

    NARCIS (Netherlands)

    Reijnders, Christianne M. A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.; Gibbs, Susan

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  10. Testosterone metabolism of fibroblasts grown from prostatic carcinoma, benign prostatic hyperplasia and skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Schweikert, H.U.; Hein, H.J.; Romijn, J.C.; Schroeder, F.H.

    1982-02-01

    The metabolism of (1,2,6,7-3H)testosterone was assessed in fibroblast monolayers derived from tissue of 5 prostates with benign hyperplasia (BPH), 4 prostates with carcinoma (PC), and 3 biopsy samples of skin, 2 nongenital skin (NG) and 1 genital skin. The following metabolites could be identified: androstanedione androstenedione, dihydrotestosterone, androsterone, epiandrosterone, androstane-3 alpha, 17 beta-diol and androstane-3 beta, 17 beta-diol. Testosterone was metabolized much more rapidly in fibroblasts originating from prostatic tissue than in fibroblasts derived from NG. A significantly higher formation of 5 alpha-androstanes and 3 alpha-hydroxysteroids could be observed in fibroblasts from BPH as compared to PC. 17-ketosteroid formation exceeded 5 alpha-androstane formation in BPH, whereas 5 alpha-reduction was the predominant pathway in fibroblasts grown from PC and NG. Since testosterone metabolism in fibroblasts of prostatic origin therefore resembles in many aspects that in whole prostatic tissue, fibroblasts grown from prostatic tissues might be a valuable tool for further investigation of the pathogenesis of human BPH and PC.

  11. Dipeptides Increase Functional Activity of Human Skin Fibroblasts.

    Science.gov (United States)

    Malinin, V V; Durnova, A O; Polyakova, V O; Kvetnoi, I M

    2015-05-01

    We analyzed the effect of dipeptide Glu-Trp and isovaleroyl-Glu-Trp in concentrations of 0.2, 2 and 20 μg/ml and Actovegin preparation on functional activity of human skin fibroblasts. Dipeptides, especially Glu-Trp, produce a stimulating effect on human skin fibroblasts and their effect is equivalent to that of Actovegin. Dipeptides stimulate cell renewal processes by activating synthesis of Ki-67 and reducing expression of caspase-9 and enhance antioxidant function of the cells by stimulating the expression of Hsp-90 and inducible NO-synthase. These findings suggest that dipeptides are promising candidates for preparations stimulating reparative processes.

  12. Primary skin fibroblasts as a model of Parkinson's disease

    NARCIS (Netherlands)

    Auburger, G.; Klinkenberg, M.; Droste, J.A.H.; Marcus, K.; Morales-Gordo, B.; Kunz, W.S.; Brandt, U.; Broccoli, V.; Reichmann, H.; Gispert, S.; Jendrach, M.

    2012-01-01

    Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts a

  13. Preclinical safety studies on autologous cultured human skin fibroblast transplantation.

    Science.gov (United States)

    Zeng, Wei; Zhang, Shuying; Liu, Dai; Chai, Mi; Wang, Jiaqi; Zhao, Yuming

    2014-01-01

    Recently, FDA approved the clinical use of autologous fibroblasts (LAVIV™) for the improvement of nasolabial fold wrinkles in adults. The use of autologous fibroblasts for the augmentation of dermal and subcutaneous defects represents a potentially exciting natural alternative to the use of other filler materials for its long-term corrective ability and absence of allergic adverse effects proved by clinical application. However, compared to the clinical evidence, preclinical studies are far from enough. In this study, human skin-derived fibroblasts were cultured and expanded for both in vitro and in vivo observations. In vitro, the subcultured fibroblasts were divided into two groups. One set of cells underwent cell cycle and karyotype analysis at passages 5 and 10. The second group of cells was cocultured in medium with different concentrations of human skin extract D for the measurement of collagen concentration and cell count. In vivo, the subcultured fibroblasts were injected into nude mice subcutaneously. Biopsies were taken for morphology observation and specific collagen staining at 1, 2, and 3 months after injection. The results in vitro showed no significant differences in cell cycle distribution between passages 5 and 10. Cell proliferation and secretion were inhibited as the concentration of extract D increased. In vivo, the fibroblasts were remarkably denser on the experimental side with no dysplastic cells. Mitotic cells were easily observed at the end of the first month but were rare at the end of the third month. Type III collagen was detected at the end of the first month, while collagen type I was positive at the end of the second month. The content of both collagens increased as time passed. The above results indicated that the use of the autologous fibroblasts was safe, providing a basic support for clinical use of fibroblasts.

  14. Skin Biopsy and Patient-Specific Stem Cell Lines

    Science.gov (United States)

    Li, Yao; Nguyen, Huy V.; Tsang, Stephen H.

    2016-01-01

    The generation of patient-specific induced pluripotent stem (iPS) cells permits the development of next-generation patient-specific systems biology models reflecting personalized genomics profiles to better understand pathophysiology. In this chapter, we describe how to create a patient-specific iPS cell line. There are three major steps: (1) performing a skin biopsy procedure on the patient; (2) extracting human fibroblast cells from the skin biopsy tissue; and (3) reprogramming patient-specific fibroblast cells into the pluripotent stem cell stage. PMID:26141312

  15. Effect of low-power red light laser irradiation on the viability of human skin fibroblast

    Energy Technology Data Exchange (ETDEWEB)

    Bednarska, K.; Rozga, B.; Leyko, W.; Bryszewska, M. [Institute of Biophysics, University of Lodz (Poland); Kolodziejczyk, K.; Szosland, D. [Diabetological Clinic, Medical Academy of Lodz (Poland)

    1998-10-01

    Human skin fibroblast monolayers (S-126 cell line) were exposed to laser radiation (wavelength 670 nm, power density 40 mW/cm{sup 2}). The energy densities were 2 J/cm{sup 2} and 12 J/cm{sup 2}, respectively, and the irradiation was carried out at a temperature of 22 C. For fibroblast viability evaluation, the colorimetric assay (conversion of thiazolyl blue to formazan) was used. The experiments were carried out at 37 C, in the presence of 5% CO{sub 2}, and at different time periods of incubation after irradiation (2, 4, 8 h and 1, 2, 3, 4, 5 days). The results indicated that there was a certain stimulating effect on the long-term proliferation of skin fibroblasts and that the stimulation proceeded in two stages, the first one 2 h and the second one 3 days post-irradiation. (orig.) With 4 figs., 2 tabs., 13 refs.

  16. Histamine inhibits differentiation of skin fibroblasts into myofibroblasts.

    Science.gov (United States)

    Lin, Lin; Yamagata, Kaoru; Nakayamada, Shingo; Sawamukai, Norifumi; Yamaoka, Kunihiro; Sakata, Kei; Nakano, Kazuhisa; Tanaka, Yoshiya

    2015-07-31

    Histamine and TGF-β, major mediators secreted by mast cells, are involved in skin inflammation and play critical roles in the pathogenesis of systemic sclerosis. However, the roles of signaling mechanisms in the development of skin fibrosis remain largely unclear. Here we show that histamine suppressed the expression of α smooth muscle actin (αSMA), a marker of myofibroblasts, induced by TGF-β1 in skin fibroblasts. Histamine H1-receptor (H1R), but not H2-receptor (H2R) or H4-receptor (H4R), was expressed on skin fibroblasts at both mRNA and protein levels. Interestingly, an H1R antagonist, but not H2R or H4R antagonists, antagonized the histamine-mediated suppression of αSMA expression by TGF-β1. Correspondingly, phosphorylated Smad2 was detected after treatment with TGF-β1, whereas the addition of histamine inhibited this phosphorylation. Taken together, histamine-H1R decreased TGF-β1-mediated Smad2 phosphorylation and inhibited differentiation of skin fibroblasts into myofibroblasts.

  17. Treatment of Skin Avulsion Injuries with Basic Fibroblast Growth Factor

    Directory of Open Access Journals (Sweden)

    Hajime Matsumine, MD, PhD

    2015-04-01

    Full Text Available Summary: This report describes favorable outcomes in 9 patients with skin avulsion injuries of the extremities who underwent full-thickness skin grafting and basic fibroblast growth factor (bFGF application. Following removal of contaminated subcutaneous fat tissue on the inside of skin, the avulsed skin was processed into a full-thickness skin graft, with as much of the skin used as possible irrespective of damage. Several drainage holes (5–10 mm in diameter were made on the graft for drainage from the graft bed and to prevent seroma and hematoma formation. Genetically recombinant human bFGF was sprayed at a dose of 1 μg/cm2 onto the graft bed, which was then covered with the graft and sutured. Pressure immobilization with ointment gauzes and elastic bandages was administered for 1 week postoperatively, and the surface of the skin grafts that did not take was scraped away, preserving the revascularized dermal component on the debrided raw surface as much as possible. bFGF was sprayed again onto the debrided surface to promote epithelialization. Wound closure was achieved in all cases with conservative therapy. The surgical procedure was effective in preventing postoperative ulcer formation and scar contracture and resulted in wound healing with the formation of good-quality, flexible scars.

  18. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  19. The in vitro photosensitivity of systemic lupus erythematosus skin fibroblasts.

    Science.gov (United States)

    Zamansky, G B; Minka, D F; Deal, C L; Hendricks, K

    1985-03-01

    To investigate the role of DNA damage in the pathogenesis of systemic lupus erythematosus (SLE), we studied the ability of skin fibroblasts derived from SLE patients to recover from ultraviolet (UV) light radiation of varying wavelengths. Four of five SLE cell strains were more sensitive to UV-C (254 nm), sun lamp, and UV-A (320 to 400 nm) light than were normal cells. SLE cellular recovery was most sensitive to broad spectrum, long wavelength light. This hypersensitivity did not appear to result from the UV light activation of a clastogenic factor. Experiments which examined the DNA repair capacity of irradiated cells indicated that SLE fibroblasts may be able to excise certain DNA lesions as well as normal cells. The mechanisms responsible for the hypersensitivity of SLE cells remain under investigation.

  20. Tattoo ink nanoparticles in skin tissue and fibroblasts.

    Science.gov (United States)

    Grant, Colin A; Twigg, Peter C; Baker, Richard; Tobin, Desmond J

    2015-01-01

    Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.

  1. Tattoo ink nanoparticles in skin tissue and fibroblasts

    Directory of Open Access Journals (Sweden)

    Colin A. Grant

    2015-05-01

    Full Text Available Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufacturers are not obliged to disclose the exact composition of their products. This study examines tattoo ink particles in two fundamental skin components at the nanometre level. We use atomic force microscopy and light microscopy to examine cryosections of tattooed skin, exploring the collagen fibril networks in the dermis that contain ink nanoparticles. Further, we culture fibroblasts in diluted tattoo ink to explore both the immediate impact of ink pigment on cell viability and also to observe the interaction between particles and the cells.

  2. Oral fibroblasts produce more HGF and KGF than skin fibroblasts in response to co-culture with keratinocytes

    DEFF Research Database (Denmark)

    Grøn, Birgitte; Stoltze, Kaj; Andersson, Anders;

    2002-01-01

    cells were maintained in collagen, the level of HGF and KGF was decreased mainly in skin cultures. However, in oral fibroblasts, induction after stimulation was at a similar level in collagen compared to on polystyrene. Skin fibroblasts maintained in collagen produced almost no HGF whether...... that could explain regional variation in epithelial growth and wound healing. Normal human fibroblasts were cultured on polystyrene or maintained in collagen matrix and stimulated with keratinocytes cultured on membranes. The amount of HGF and KGF protein in the culture medium was determined every 24 h for 5...

  3. Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

    Science.gov (United States)

    Yamaba, Hiroyuki; Haba, Manami; Kunita, Mayumi; Sakaida, Tsutomu; Tanaka, Hiroshi; Yashiro, Youichi; Nakata, Satoru

    2016-08-01

    Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.

  4. Exposure to Varying Strain Magnitudes Influences the Conversion of Normal Skin Fibroblasts Into Hypertrophic Scar Cells.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Cai, Xia; Liu, Tao

    2016-04-01

    Mechanical strain is a key contributor in the pathogenesis of hypertrophic scarring, whose optimal stretch magnitudes to initiate the differentiation of normal skin fibroblasts into aberrant fibroblasts phenotype remains largely unresolved. Influence of varying cyclic strain magnitudes on cultured human normal skin fibroblasts and its transformation into hypertrophic scar fibroblast-like phenotype is investigated in this study. Cultured fibroblasts isolated from hypertrophic scar and normal skin tissue were subjected to cyclic mechanical stretching under individual 10%, 15%, and 20% strain magnitudes at a frequency of 0.1 Hz for 24 hours. Stretched normal skin fibroblasts demonstrated significantly increased rates of cell proliferation, and also apparently oriented away nearly perpendicular to the applied stretching direction. Interestingly, the applied 10% strains magnitude resulted in a markedly enhanced cell proliferative ability compared with that of 20% strain magnitude. Parameters involving the mechanotransduction signaling, such as integrin β1 and P130Cas, were significantly improved at both mRNA and protein levels in the stretched normal skin fibroblasts, which was demonstrated in a negative magnitude-dependent manner. In addition, 10% strains magnitude triggered the highest expression levels of growth factor TGF-β1 and collagen matrix in stretched normal skin fibroblasts. Collectively, these results indicate that the 10% stretching magnitude, of the 3 strain magnitudes studied, is most effective for triggering the optimal mechanotransduction effects and biological responses inside cultured skin fibroblasts. The demonstrable conversion of normal skin fibroblasts into hypertrophic scar fibroblasts was also observed when 10% stretching magnitude was applied to cultured fibroblasts in vitro.

  5. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

    Directory of Open Access Journals (Sweden)

    Emond Mary

    2007-09-01

    Full Text Available Abstract Background Marfan syndrome (MFS is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value -6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status. An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater.

  6. A Marfan syndrome gene expression phenotype in cultured skin fibroblasts

    Science.gov (United States)

    Yao, Zizhen; Jaeger, Jochen C; Ruzzo, Walter L; Morale, Cecile Z; Emond, Mary; Francke, Uta; Milewicz, Dianna M; Schwartz, Stephen M; Mulvihill, Eileen R

    2007-01-01

    Background Marfan syndrome (MFS) is a heritable connective tissue disorder caused by mutations in the fibrillin-1 gene. This syndrome constitutes a significant identifiable subtype of aortic aneurysmal disease, accounting for over 5% of ascending and thoracic aortic aneurysms. Results We used spotted membrane DNA macroarrays to identify genes whose altered expression levels may contribute to the phenotype of the disease. Our analysis of 4132 genes identified a subset with significant expression differences between skin fibroblast cultures from unaffected controls versus cultures from affected individuals with known fibrillin-1 mutations. Subsequently, 10 genes were chosen for validation by quantitative RT-PCR. Conclusion Differential expression of many of the validated genes was associated with MFS samples when an additional group of unaffected and MFS affected subjects were analyzed (p-value < 3 × 10-6 under the null hypothesis that expression levels in cultured fibroblasts are unaffected by MFS status). An unexpected observation was the range of individual gene expression. In unaffected control subjects, expression ranges exceeding 10 fold were seen in many of the genes selected for qRT-PCR validation. The variation in expression in the MFS affected subjects was even greater. PMID:17850668

  7. Mercury-induced micronuclei in skin fibroblasts of beluga whales

    Energy Technology Data Exchange (ETDEWEB)

    Gauthier, J.M.; Dubeau, H.; Rassart, E. [Univ. du Quebec, Montreal, Quebec (Canada). Dept. des Sciences Biologiques

    1998-12-01

    Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high incidence of cancer observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei assay was used to test the genotoxic potential of mercury compounds in skin fibroblasts of an Arctic beluga whale. Both mercuric chloride (Hg) and methylmercury (MeHg) induced a highly significant dose-response increase of micronucleated cells. Statistically significant increases in micronucleated cells were observed for 0.5, 5, and 20 {micro}g/ml Hg and 0.05, 0.5, and 2 {micro}g/ml MeHg when compared to control cultures. Concentrations of 0.5, 5, and 20 {micro}g/ml Hg induced a two-, three- and fourfold increase of micronucleated cells, respectively. Treatment with MeHg was one order of magnitude more potent in inducing micronuclei and in inhibiting cell proliferation than Hg. Although results of this in vitro study do not imply that mercury compounds are involved in the etiology of cancer in St. Lawrence beluga whales, significant increases in micronuclei frequency were found at low concentrations of MeHg that are believed to be comparable to concentrations present in certain whales of this population.

  8. PTCH1 +/- dermal fibroblasts isolated from healthy skin of Gorlin syndrome patients exhibit features of carcinoma associated fibroblasts.

    Directory of Open Access Journals (Sweden)

    Alexandre Valin

    Full Text Available Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS causes predisposition to basal cell carcinoma (BCC, the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3 and NBCCS fibroblasts bearing either nonsense (n = 3 or missense (n = 3 PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1(+/- genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients.

  9. Sildenafil attenuates the fibrotic phenotype of skin fibroblasts in patients with systemic sclerosis.

    Science.gov (United States)

    Higuchi, Tomoaki; Kawaguchi, Yasushi; Takagi, Kae; Tochimoto, Akiko; Ota, Yuko; Katsumata, Yasuhiro; Ichida, Hisae; Hanaoka, Masanori; Kawasumi, Hidenaga; Tochihara, Mari; Yamanaka, Hisashi

    2015-12-01

    Systemic sclerosis (SSc) is a multi-organ fibrotic disease that affects the skin and various internal organs. Therapeutic strategies for tissue fibrosis have not been established; however, aberrantly activated fibroblasts in affected lesions are key targets for modulating fibrosis. Recently, increased intracellular cyclic GMP (cGMP) levels were demonstrated to improve fibrosis levels in various diseases. The purpose of this study was to assess the anti-fibrotic properties of cGMP in cultured fibroblasts from patients with SSc. The phosphodiesterase (PDE) 5 inhibitor sildenafil increased the intracellular cGMP levels in skin fibroblasts in a dose-dependent manner. Sildenafil treatment also significantly decreased the expression of several pro-fibrotic factors that were upregulated by TGF-β1 treatment in SSc skin fibroblasts. These inhibitory effects occurred via non-canonical TGF-β signaling. Our findings revealed that sildenafil might be a novel strategy to treat tissue fibrosis and vasculopathy in SSc.

  10. Cloned goats (Gapra hircus) from foetal fibroblast cell lines

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Mammalian cloning has been one of the most active research topics in the world.Cloning with in vitro culured foetal fibroblast cells,in comparison with embryonic cells,can be used not only to theoretically study the embryonic or cellular development and differentiation in mammals,but also to utilize the unlimited fibroblast cells to produce large numbers of clonings.The preliminary results are as follows:(i) The division and development of the cloned embryos with embryonic donor cells and goat foetal fibroblast donor cells were 55%,77% and 35%,31%,respectively.There is no significant statistical difference between them.(ii) These studies result in the birth of two cloned goats derived from two 30-day foetal fibroblast cell lines,which are the first cloned mammals from somatic cells in China.This project has established a technological data base for the furture research on adult mammalian somatic cloning and nucleocytoplasmic interactions in animal development,and a novel technique for the cloning of animals with a high-level expression of transgene(s).

  11. Matrine inhibits proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB

    Institute of Scientific and Technical Information of China (English)

    WU Yan-an; GAO Chun-fang; WANG Hao; HUANG Chao; KONG Xian-tao

    2001-01-01

    To study the effect of matrine on proliferation of mouse skin fibroblasts induced by platelet-derived growth factor-BB (PDGF-BB). Methods: Mouse skin fibroblasts were obtained from newborn ⅠCR mice and propagated in vitro. Proliferation of cell was analyzed by mitochondrial reduction of tetrazolium salt MTT and actual cell count. Results: Matrine (50 to 500 μg/ml) caused dose-dependent reduction of serum-stimulated cell growth. Growth inhibition was totally reversed after removal of the drug. Matrine also inhibited PDGF-BB induced cell growth dose-dependently. Conclusion: Matrine exhibits potent anti-proliferation effect on mouse skin fibroblast. This effect appears to be mediated by decrease of PDGF-induced growth. These results suggest that matrine might have preventive and therapeutic implication in skin fibrosis.

  12. Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts.

    Science.gov (United States)

    Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

    2017-01-01

    Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine(9) (pGSK3β Ser(9)) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts.

  13. Fibroblast heterogeneity and its implications for engineering organotypic skin models in vitro.

    Science.gov (United States)

    Sriram, Gopu; Bigliardi, Paul Lorenz; Bigliardi-Qi, Mei

    2015-11-01

    Advances in cell culture methods, multidisciplinary research, clinical need to replace lost skin tissues and regulatory need to replace animal models with alternative test methods has led to development of three dimensional models of human skin. In general, these in vitro models of skin consist of keratinocytes cultured over fibroblast-populated dermal matrices. Accumulating evidences indicate that mesenchyme-derived signals are essential for epidermal morphogenesis, homeostasis and differentiation. Various studies show that fibroblasts isolated from different tissues in the body are dynamic in nature and are morphologically and functionally heterogeneous subpopulations. Further, these differences seem to be dictated by the local biological and physical microenvironment the fibroblasts reside resulting in "positional identity or memory". Furthermore, the heterogeneity among the fibroblasts play a critical role in scarless wound healing and complete restoration of native tissue architecture in fetus and oral mucosa; and excessive scar formation in diseased states like keloids and hypertrophic scars. In this review, we summarize current concepts about the heterogeneity among fibroblasts and their role in various wound healing environments. Further, we contemplate how the insights on fibroblast heterogeneity could be applied for the development of next generation organotypic skin models.

  14. Aging alters functionally human dermal papillary fibroblasts but not reticular fibroblasts: a new view of skin morphogenesis and aging.

    Directory of Open Access Journals (Sweden)

    Solène Mine

    Full Text Available Understanding the contribution of the dermis in skin aging is a key question, since this tissue is particularly important for skin integrity, and because its properties can affect the epidermis. Characteristics of matched pairs of dermal papillary and reticular fibroblasts (Fp and Fr were investigated throughout aging, comparing morphology, secretion of cytokines, MMPs/TIMPs, growth potential, and interaction with epidermal keratinocytes. We observed that Fp populations were characterized by a higher proportion of small cells with low granularity and a higher growth potential than Fr populations. However, these differences became less marked with increasing age of donors. Aging was also associated with changes in the secretion activity of both Fp and Fr. Using a reconstructed skin model, we evidenced that Fp and Fr cells do not possess equivalent capacities to sustain keratinopoiesis. Comparing Fp and Fr from young donors, we noticed that dermal equivalents containing Fp were more potent to promote epidermal morphogenesis than those containing Fr. These data emphasize the complexity of dermal fibroblast biology and document the specific functional properties of Fp and Fr. Our results suggest a new model of skin aging in which marked alterations of Fp may affect the histological characteristics of skin.

  15. Establishment of the first humpback whale fibroblast cell lines and their application in chemical risk assessment

    Energy Technology Data Exchange (ETDEWEB)

    Burkard, Michael, E-mail: Michael.burkard@eawag.ch [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia); Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); Whitworth, Deanne [The University of Queensland, School of Veterinary Science, Gatton, QLD (Australia); Schirmer, Kristin [Eawag, Swiss Federal Institute of Technology, Dübendorf (Switzerland); ETH Zürich, Institute of Biogechemistry and Pollutant Dynamics, Zürich (Switzerland); EPF Lausanne, School of Architecture, Civil and Environmental Engineering, Lausanne (Switzerland); Nash, Susan Bengtson [Griffith University, Environmental Futures Research Institute, Southern Ocean Persistent Organic Pollutants Program, Brisbane, QLD (Australia)

    2015-10-15

    Highlights: • We established and characterised the first humpback whale fibroblast cell lines. • Cell lines have a stable karyotype with 2n = 44. • Exposure to p,p′-DDE resulted in a concentration-dependent loss of cell viability. • p,p′-DDE sensitivity differed considerably from human fibroblasts. • Exposure to a whale blubber extract showed higher sensitivity than to p,p′-DDE alone. - Abstract: This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO{sub 2} in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n = 44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para′-dichlorodiphenyldichloroethylene (p,p′-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC{sub 50} value) was approximately six times greater than the EC{sub 50} value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p′-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p′-DDE alone

  16. Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging.

    Directory of Open Access Journals (Sweden)

    Christine Duval

    Full Text Available To study cutaneous pigmentation in a physiological context, we have previously developed a functional pigmented reconstructed skin model composed of a melanocyte-containing epidermis grown on a dermal equivalent comprising living fibroblasts. The present studies, using the same model, aimed to demonstrate that dermal fibroblasts influence skin pigmentation up to the macroscopic level. The proof of principle was performed with pigmented skins differing only in the fibroblast component. First, the in vitro system was reconstructed with or without fibroblasts in order to test the global influence of the presence of this cell type. We then assessed the impact of the origin of the fibroblast strain on the degree of pigmentation using fetal versus adult fibroblasts. In both experiments, impressive variation in skin pigmentation at the macroscopic level was observed and confirmed by quantitative parameters related to skin color, melanin content and melanocyte numbers. These data confirmed the responsiveness of the model and demonstrated that dermal fibroblasts do indeed impact the degree of skin pigmentation. We then hypothesized that a physiological state associated with pigmentary alterations such as photo-aging could be linked to dermal fibroblasts modifications that accumulate over time. Pigmentation of skin reconstructed using young unexposed fibroblasts (n = 3 was compared to that of tissues containing natural photo-aged fibroblasts (n = 3 which express a senescent phenotype. A stimulation of pigmentation in the presence of the natural photo-aged fibroblasts was revealed by a significant increase in the skin color (decrease in Luminance and an increase in both epidermal melanin content and melanogenic gene expression, thus confirming our hypothesis. Altogether, these data demonstrate that the level of pigmentation of the skin model is influenced by dermal fibroblasts and that natural photo-aged fibroblasts can contribute to the

  17. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts

    DEFF Research Database (Denmark)

    Noël, D; Pelegrin, M; Brockly, F;

    2000-01-01

    In vivo production and systemic delivery of therapeutic antibodies by engineered cells might advantageously replace injection of purified antibodies for treating a variety of life-threatening diseases, including cancer, acquired immunodeficiency syndrome, and autoimmune diseases. We report here...... that skin fibroblasts retrovirally transduced to express immunoglobulin genes can be used for sustained long-term systemic delivery of cloned antibodies in immunocompetent mice. Importantly, no anti- idiotypic response against the ectopically expressed model antibody used in this study was observed....... This supports the notion that skin fibroblasts can potentially be used in antibody-based gene/cell therapy protocols without inducing any adverse immune response in treated individuals....

  18. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

    NARCIS (Netherlands)

    El Ghalbzouri, A; Jonkman, MF; Dijkman, R; Ponec, M

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence

  19. Neurotensin Decreases the Proinflammatory Status of Human Skin Fibroblasts and Increases Epidermal Growth Factor Expression

    Directory of Open Access Journals (Sweden)

    Lucília Pereira da Silva

    2014-01-01

    Full Text Available Fibroblasts colonization into injured areas during wound healing (WH is responsible for skin remodelling and is also involved in the modulation of inflammation, as fibroblasts are immunologically active. Herein, we aimed to determine neurotensin effect on the immunomodulatory profile of fibroblasts, both in homeostatic and inflammatory conditions. Neurotensin mediated responses occurred through NTR1 or NTR3 receptors, while under inflammatory conditions NTR1 expression increase seemed to modulate neurotensin responses. Among different immunomodulatory genes, CCL11, IL-8, and IL-6 were the most expressed genes, while CCL4 and EGF were the less expressed genes. After neurotensin exposure, IL-8 mRNA expression was increased while CCL11 was decreased, suggesting a proinflammatory upregulation and chemoattractant ability downregulation of fibroblasts. Under inflammatory conditions, gene expression was significantly increased. After neurotensin exposure, CCL4 and IL-6 mRNA expression were decreased while CCL11 was increased, suggesting again a decrease in the chemoattractant capacity of fibroblasts and in their proinflammatory status. Furthermore, the expression of EGF, a crucial growth factor for skin cells proliferation and WH, was increased in all conditions. Overall, neurotensin, released by nerve fibers or skin cells, may be involved in the decrease of the chemotaxis and the proinflammatory status in the proliferation and remodelling phases of WH.

  20. Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

    Science.gov (United States)

    Demirovic, Dino; Nizard, Carine; Rattan, Suresh I S

    2015-01-01

    Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.

  1. Cyclical cell stretching of skin-derived fibroblasts downregulates connective tissue growth factor (CTGF) production.

    Science.gov (United States)

    Kanazawa, Yuichiro; Nomura, Jun; Yoshimoto, Shinya; Suzuki, Toshikazu; Kita, Kazuko; Suzuki, Nobuo; Ichinose, Masaharu

    2009-01-01

    Delayed healing of skin wounds can be caused by wound instability, whereas appropriate massage or exercise prevents sclerosis and scar contracture. However, the mechanism by which wound healing is related to mechanical stress has not been fully elucidated. The present study aimed to identify whether mechanical stretching of fibroblasts reduces their production of extracellular matrix. We transferred skin fibroblasts into collagen-coated elastic silicone chambers, cultured them on a stretching apparatus, and used RT-PCR to examine the effects of mechanical stretching on the expression levels of 17 genes related to extracellular matrix production and growth factor secretion. We found that connective tissue growth factor (CTGF) was downregulated after 24 hr of cell stretching. Specifically, the CTGF mRNA and protein levels were 50% and 48% of the control levels, respectively. These findings suggest that cyclic stretching of fibroblasts contributes to anti-fibrotic processes by reducing CTGF production.

  2. ADHESION AND SPREADING OF HUMAN SKIN FIBROBLASTS ON PHYSICOCHEMICALLY CHARACTERIZED GRADIENT SURFACES

    NARCIS (Netherlands)

    RUARDY, TG; SCHAKENRAAD, JM; VANDERMEI, HC; BUSSCHER, HJ

    1995-01-01

    In this study, adhesion and spreading of human skin fibroblasts on gradient surfaces of dichlorodimethylsilane (DDS) coupled to glass was investigated. Gradient surfaces were prepared by the diffusion technique and characterized by the Wilhelmy plate technique for their wettability and by scanning x

  3. Superficial dermal fibroblasts enhance basement membrane and epidermal barrier formation in tissue-engineered skin: implications for treatment of skin basement membrane disorders.

    Science.gov (United States)

    Varkey, Mathew; Ding, Jie; Tredget, Edward E

    2014-02-01

    Basement membrane is a highly specialized structure that binds the dermis and the epidermis of the skin, and is mainly composed of laminins, nidogen, collagen types IV and VII, and the proteoglycans, collagen type XVIII and perlecan, all of which play critical roles in the function and resilience of skin. Both dermal fibroblasts and epidermal keratinocytes contribute to the development of the basement membrane, and in turn the basement membrane and underlying dermis influence the development and function of the epidermal barrier. Disruption of the basement membrane results in skin fragility, extensive painful blistering, and severe recurring wounds as seen in skin basement membrane disorders such as epidermolysis bullosa, a family of life-threatening congenital skin disorders. Currently, there are no successful strategies for treatment of these disorders; we propose the use of tissue-engineered skin as a promising approach for effective wound coverage and to enhance healing. Fibroblasts and keratinocytes isolated from superficial and deep dermis and epidermis, respectively, of tissue from abdominoplasty patients were independently cocultured on collagen-glycosaminoglycan matrices, and the resulting tissue-engineered skin was assessed for functional differences based on the underlying specific dermal fibroblast subpopulation. Tissue-engineered skin with superficial fibroblasts and keratinocytes formed a continuous epidermis with increased epidermal barrier function and expressed higher levels of epidermal proteins, keratin-5, and E-cadherin, compared to that with deep fibroblasts and keratinocytes, which had an intermittent epidermis. Further, tissue-engineered skin with superficial fibroblasts and keratinocytes formed better basement membrane, and produced more laminin-5, nidogen, collagen type VII, compared to that with deep fibroblasts and keratinocytes. Overall, our results demonstrate that tissue-engineered skin with superficial fibroblasts and keratinocytes

  4. Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts.

    Science.gov (United States)

    Opländer, Christian; Wetzel, Wiebke; Cortese, Miriam M; Pallua, Norbert; Suschek, Christoph V

    2008-05-01

    Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.

  5. Ontogeny of expression of basic fibroblast growth factor and its receptors in human fetal skin

    Institute of Scientific and Technical Information of China (English)

    CHEN Wei; FU Xiao-bing; GE Shi-li; SUN Tong-zhu; SHENG Zhi-yong

    2005-01-01

    Objective : To investigate the expression characteristics of basic fibroblast growth factor (bFGF)and its receptors, flg ( FGFR1 ) and bek ( FGFR2), in fetal skin at different gestational ages underlying the relevance of these 3 proteins to skin development and the mechanisms underlying the phenotypic transition from scarless to scarforming healing.Methods: Eighteen specimens of fetal skin biopsies of human embryo were obtained from spontaneous abortions at different gestational ages of 13-32 weeks. Gene expression of bFGF, bek and flg was examined with reverse transcription-polymerase chain reaction (RT-PCR). The dynamic expression and distribution of these 3 proteins were detected with streptavidin peroxidase ( SP )immunohistochemical staining method.Results: In the early gestational fetal skin, genes of bFGF and flg were strongly expressed and more protein contents of these 2 proteins were found as compared with the genes at late gestation fetal skin (2.446 ± 0.116 and 2.066 ± 0. 152 versus 2.157 ± 0. 101 and 1.818 ± 0.086,respectively, P < 0.05). On the contrary, the levels of gene expression and protein content of bek were not differently expressed in the early gestational fetal skin versus the late ones. Protein particles of bFGF were mainly distributed in the epidermal cells and some fibroblasts. Bek was mainly located in the cell membrane and cytoplasm of epidermal cells while flg protein was principally located in the epidermal cells, endothelial cells and some fibroblasts.Conclusions: The endogenous bFGF and their receptors might be involved in the cutaneous development at fetal stage. The differently expressing levels of bFGF and flg during gestation may be related to scarless or scarforming repair during gestation.

  6. Development and validation of a skin fibroblast biomarker profile for schizophrenic patients

    Directory of Open Access Journals (Sweden)

    Marianthi Logotheti

    2016-12-01

    Full Text Available Gene expression profiles of non-neural tissues through microarray technology could be used in schizophrenia studies, adding more information to the results from similar studies on postmortem brain tissue. The ultimate goal of such studies is to develop accessible biomarkers. Supervised machine learning methodologies were used, in order to examine if the gene expression from skin fibroblast cells could be exploited for the classification of schizophrenic subjects. A dataset of skin fibroblasts gene expression of schizophrenia patients was obtained from Gene Expression Omnibus database. After applying statistical criteria, we concluded to genes that present a differential expression between the schizophrenic patients and the healthy controls. Based on those genes, functional profiling was performed with the BioInfoMiner web tool. After the statistical analysis, 63 genes were identified as differentially expressed. The functional profiling revealed interesting terms and pathways, such as mitogen activated protein kinase and cyclic adenosine monophosphate signaling pathways, as well as immune-related mechanisms. A subset of 16 differentially expressed genes from fibroblast gene expression profiling that occurred after Support Vector Machines Recursive Feature Elimination could efficiently separate schizophrenic from healthy controls subjects. These findings suggest that through the analysis of fibroblast based gene expression signature and with the application of machine learning methodologies we might conclude to a diagnostic classification model in schizophrenia.

  7. Assembly of fibronectin into the extracellular matrix of early and late passage human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Mann, D.M.

    1987-01-01

    The specific binding of soluble /sup 125/I-human plasma fibronectin (/sup 125/I-HFN-P) to confluent cultures of early and late passage human skin fibroblasts was investigated. Previous studies HFN-P bound to fibroblast cell layers indicated that HNF-P was present in the cultures in two separate pools, distinguishable on the basis of their solubility in 1% deoxycholate. Examination of the kinetics of /sup 125/I-HFN-P binding to Pool I of early and late passage cultures revealed that both cultures required 2-4 h to approach steady-state conditions. Other kinetic studies showed that the rates of low of /sup 125/I-HFN-P from either Pool I or Pool II were similar for both cultures. Further, Scatchard analysis revealed a single class of Pool I binding sites with apparent dissociation constants (K/sub d/) of 5.3 x 10/sup -8/M (early passage) and 4.2 x 10/sup -8/M (late passage). These results indicate that early and late passage cultures of human fibroblasts exhibit differences in the number of cell surface biding sites for soluble fibronectin, and in the extent to which they incorporate soluble fibronectin into the extracellular matrix. Parameters which affect the fibronectin matrix assembly system of human skin fibroblasts were also examined. In addition, several monoclonal anti-fibronectin antibodies were characterized and developed as experimental probes for fibronectin structure and function.

  8. DNA damage in human skin fibroblasts exposed to UVA light used in clinical PUVA treatment

    Energy Technology Data Exchange (ETDEWEB)

    Bredberg, A.

    1981-06-01

    Human skin fibroblasts were irradiated with a clinically used UVA light source. The doses (1.1 and 3 J/cm2) were similar to those reaching the dermis during clinical PUVA treatment of psoriasis. DNA strand breaks, as determined by alkaline elution, were formed in a dose-dependent way and disappeared within 1 hr of postincubation at 37 degrees C. These findings have clinical implications since UVA-induced DNA damage may be accompanied by mutagenic and tumor promoting effects.

  9. Juglans mandshurica leaf extract protects skin fibroblasts from damage by regulating the oxidative defense system.

    Science.gov (United States)

    Park, Gunhyuk; Jang, Dae Sik; Oh, Myung Sook

    2012-05-01

    Skin is mainly damaged by genetic and environmental factors such as ultraviolet light, xenobiotics, hormonal changes, heat, and smoking. ROS production is commonly involved in the pathogenesis of skin damage induced by these factors, causing skin aging, including wrinkling, by activating the metalloproteinases (MMP-1) that break down type I collagen (COL1A1). The walnut tree Juglans mandshurica MAX. (JM) is found in China, Siberia and Korea. JM has been reported to have various pharmacological activities, such as anti-tumor, anti-oxidative, and anti-bacterial effects. In the present study, we investigated the protective effect of JM leaf extract (JME) against oxidative stress in HS68 human skin fibroblasts. JME significantly and dose-dependently protected HS68 cells against H₂O₂-induced damage, as assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay. Other assays demonstrated that JME protected HS68 cells by regulating ROS production and increasing levels of glutathione, heme oxygenase-1, and activated NF-E2-related factor 2. JME additionally prevented the elevation of MMP-1 and reduction of COL1A1 induced by H₂O₂. It also inhibited H₂O₂-induced phosphorylation of ERK, p38, and JNK. These results indicate that JME protects human skin fibroblasts from H₂O₂-induced damage by regulating the oxidative defense system.

  10. Dimethylarsenic acid damages cellular DNA and inhibits gap junctional intercellular communication between human skin fibroblast cells

    Institute of Scientific and Technical Information of China (English)

    GuoXB; DengFR

    2002-01-01

    Although arsenic is identified as a human carcinogen,there is currently no accepted mechanism for its action or an established animal model for evaluating the carcinogenic activity of arsenic.To elucidate the mechanism of arsenic arcinogenesis,we investigated the effect of dimethylarsenic acid(DMAA),the main metabolite of inorganic arsenic in humans,on the cellular DNA and gap junctional intercellular communication (GJIC) between human skin fibroblast cells.Single-cell gel electrophoresis (SCGE) assay was used to detect the DNA damage in human skin fibroblast cells exposed to DMAA,and the GJIC between cells was detected by the scrape loading/dye transfer assay.DMAA at concentrations of 0.01-1.0 mmol·L-1 induced DNA damage in a dose-dependent manner,and GJIC between human skin fibroblast cells was significantly inhibited by DMAA at 1.0 mmol·L-1.Our results suggest that both genotoxic and nongenotoxic mechanism are involved in the mechanism of DMAA-induced cellular toxicity.

  11. The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

    Science.gov (United States)

    Saini, Natalie; Chan, Kin; Grimm, Sara A.; Dai, Shuangshuang; Fargo, David C.; Kaufmann, William K.; Taylor, Jack A.; Lee, Eunjung; Cortes-Ciriano, Isidro; Park, Peter J.; Schurman, Shepherd H.; Malc, Ewa P.; Mieczkowski, Piotr A.

    2016-01-01

    Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307 PMID:27788131

  12. Influence of hyperthermia on the phosphorylation of ribosomal protein S6 from human skin fibroblasts and meningioma cells

    DEFF Research Database (Denmark)

    Richter, W W; Zang, K D; Issinger, O G

    1983-01-01

    Skin fibroblasts and meningioma cells, derived from primary cultures of the same patients have been used to study the influence of hyperthermia on (i) cell morphology and (ii) phosphorylation pattern of ribosomal and ribosome-associated proteins. Incubation of tumour cells and fibroblasts up to 7 h...

  13. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    OpenAIRE

    2013-01-01

    PURPOSE: Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formation and stratification in a humanized animal model. METHODS: Dermo-epidermal skin grafts with either amniocytes or with fibroblasts in the dermis were compared in a rat model. Full-thicknes...

  14. Characterization of Skin Aging-Associated Secreted Proteins (SAASP) Produced by Dermal Fibroblasts Isolated from Intrinsically Aged Human Skin.

    Science.gov (United States)

    Waldera Lupa, Daniel M; Kalfalah, Faiza; Safferling, Kai; Boukamp, Petra; Poschmann, Gereon; Volpi, Elena; Götz-Rösch, Christine; Bernerd, Francoise; Haag, Laura; Huebenthal, Ulrike; Fritsche, Ellen; Boege, Fritz; Grabe, Niels; Tigges, Julia; Stühler, Kai; Krutmann, Jean

    2015-08-01

    Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.

  15. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Science.gov (United States)

    Donejko, Magdalena; Rysiak, Edyta; Galicka, Elżbieta; Terlikowski, Robert; Głażewska, Edyta Katarzyna; Przylipiak, Andrzej

    2017-01-01

    Aim The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA) on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK), and the influence of HA on those processes. Materials and methods Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. PMID:28293103

  16. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-02-01

    The authors evaluated the interaction of (/sub 3/H)1,25(OH)/sup 2/D3 with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)/sub 2/D (1,25(OH)/sub 2/D). They analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer. With normal fibroblasts the affinity and capacity of nuclear uptake of (/sub 3/H)1,25(OH)/sup 2/D3 were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. The following four patterns of interaction with (/sub 3/H)1,25(OH)/sup 2/D3 were observed with cells cultured from affected patients. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of (/sub 3/H)1,25(OH)/sup 2/D3 that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH)/sup 2/D resulted from five or six distinct genetic mutations in six kindreds.

  17. Resistance to 1,25-dihydroxyvitamin D. Association with heterogeneous defects in cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Liberman, U.A.; Eil, C.; Marx, S.J.

    1983-02-01

    We evaluated the interaction of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ with skin fibroblasts cultured from normal subjects or from affected members of six kindreds with rickets and resistance to 1-alpha, 25(OH)/sub 2/D (1,25(OH)/sub 2/D). We analyzed two aspects of the radioligand interaction; nuclear uptake with dispersed, intact cells at 37 degrees C and binding at 0 degrees C with soluble extract (cytosol) prepared from cells disrupted in buffer containing 300 mM KCl and 10 mM sodium molybdate. With normal fibroblasts the affinity and capacity of nuclear uptake of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ were 0.5 nM and 10,300 sites per cell, respectively; for binding with cytosol these were 0.13 nM and 8,900 sites per cell, respectively. In all cases where the radioligand bound with high affinity in nucleus or cytosol, the nucleus- or cytosol-associated radioligand exhibited normal sedimentation velocity on sucrose density gradients. When two kindreds exhibited similar patterns (i.e. pattern a or c) with the analyses of cultured fibroblasts, clinical features in affected members suggested that the underlying genetic defects were not identical. In conclusion: (a) Fibroblasts cultured from human skin manifest nuclear uptake and cytosol binding of (/sup 3/H)1,25(OH)/sub 2/D/sub 3/ that is an expression of the genes determining these processes in target tissues. (b) Based upon data from clinical evaluations and from analyses of cultured fibroblasts, severe resistance to 1,25(OH)/sub 2/D resulted from five or six distinct genetic mutations in six kindreds.

  18. Preventive effects of tamarind seed coat extract on UVA-induced alterations in human skin fibroblasts.

    Science.gov (United States)

    Phetdee, Khemjira; Rakchai, Racharat; Rattanamanee, Kwanchai; Teaktong, Thanasak; Viyoch, Jarupa

    2014-01-01

    One of the most damaging actions on skin is from solar radiation, particularly from its ultraviolet (UV) component, through the formation of oxidative species. Thus, an antioxidant strategy that prevents the formation of these oxidants could form the basis of an efficacious cutaneous protectant. Many herbal materials contain antioxidant polyphenols, and this study assessed the possibility that tamarind seed coat extract could fulfill this role. An alcoholic extract of the tamarind (Tamarindus indica L.) seed coat showed stronger antioxidant activity (2,2-diphenyl-1-picrylhydrazyl inhibition, EC(50) = 12.9 μg/ml) than L-ascorbic acid (EC(50) = 22.9 μg/ml) and α-tocopherol (EC(50) = 29.3 μg/ml). In cultured fibroblasts taken from human skin, hydrogen peroxide (100-1000 μM) damaged 62-92% of the cells compared to only 35-47% when the cells were preincubated in extract (200 μg/ml) for 24 h. UVA (40 J/cm2) irradiation of human fibroblasts damaged 25% of the cells but the death rate was reduced to 10% with extract. UV irradiation increased the proportion of cells arrest in G(0)/G(1) phase (from 59% to 78%) but this was largely prevented by the extract (64%), according to flow cytometry. Intracellular total glutathione of UVA-irradiated cells pretreated with the extract increased to 10-25% compared to the non-pretreated group at 24-72 h after irradiation. Fibroblasts typically increased matrix metalloproteinase-1 secretion after photodamage, and this is prevented by the extract. This is the first report showing that tamarind seed coat extract is an antioxidant and can protect human skin fibroblasts from cellular damage produced by UVA and thus may form the foundation for an antiaging cosmetic.

  19. Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

    Science.gov (United States)

    Amabile, Sonia; Belmonte, Giuseppe; Valacchi, Giuseppe; Galano, Jean-Marie; Ciccoli, Lucia; Renieri, Alessandra; Hayek, Joussef

    2014-01-01

    Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT), a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16) we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs), F4-Neuroprostanes (F4-NeuroPs), nonprotein bound iron (NPBI), and (4-HNE PAs), and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds), F2-IsoPs (7.5-folds) NPBI (2.3-folds), 4-HNE PAs (1.48-folds), and GSSG (1.44-folds) were detected, with significantly decreased GSH (−43.6%) and GSH/GSSG ratio (−3.05 folds). A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients. PMID:24987493

  20. Redox Imbalance and Morphological Changes in Skin Fibroblasts in Typical Rett Syndrome

    Directory of Open Access Journals (Sweden)

    Cinzia Signorini

    2014-01-01

    Full Text Available Evidence of oxidative stress has been reported in the blood of patients with Rett syndrome (RTT, a neurodevelopmental disorder mainly caused by mutations in the gene encoding the Methyl-CpG-binding protein 2. Little is known regarding the redox status in RTT cellular systems and its relationship with the morphological phenotype. In RTT patients (n = 16 we investigated four different oxidative stress markers, F2-Isoprostanes (F2-IsoPs, F4-Neuroprostanes (F4-NeuroPs, nonprotein bound iron (NPBI, and (4-HNE PAs, and glutathione in one of the most accessible cells, that is, skin fibroblasts, and searched for possible changes in cellular/intracellular structure and qualitative modifications of synthesized collagen. Significantly increased F4-NeuroPs (12-folds, F2-IsoPs (7.5-folds NPBI (2.3-folds, 4-HNE PAs (1.48-folds, and GSSG (1.44-folds were detected, with significantly decreased GSH (−43.6% and GSH/GSSG ratio (−3.05 folds. A marked dilation of the rough endoplasmic reticulum cisternae, associated with several cytoplasmic multilamellar bodies, was detectable in RTT fibroblasts. Colocalization of collagen I and collagen III, as well as the percentage of type I collagen as derived by semiquantitative immunofluorescence staining analyses, appears to be significantly reduced in RTT cells. Our findings indicate the presence of a redox imbalance and previously unrecognized morphological skin fibroblast abnormalities in RTT patients.

  1. Mechanism of choline deficiency and membrane alteration in postural orthostatic tachycardia syndrome primary skin fibroblasts.

    Science.gov (United States)

    Schenkel, Laila C; Singh, Ratnesh K; Michel, Vera; Zeisel, Steven H; da Costa, Kerry-Ann; Johnson, Amy R; Mudd, Harvey S; Bakovic, Marica

    2015-05-01

    Fibroblasts from a patient with postural orthostatic tachycardia syndrome (POTS), who presented with low plasma choline and betaine, were studied to determine the metabolic characteristics of the choline deficiency. Choline is required for the synthesis of the phospholipid phosphatidylcholine (PC) and for betaine, an important osmoregulator. Here, choline transport, lipid homeostasis, and mitochondria function were analyzed in skin fibroblasts from POTS and compared with control cells. The choline transporter-like protein 1/solute carrier 44A1 (CTL1/SLC44A1) and mRNA expression were 2-3 times lower in POTS fibroblasts, and choline uptake was reduced 60% (P < 0.05). Disturbances of membrane homeostasis were observed by reduced ratios between PC:phosphatidylethanolamine and sphingomyelin:cholesterol, as well as by modified phospholipid fatty acid composition. Choline deficiency also impaired mitochondria function, which was observed by a reduction in oxygen consumption, mitochondrial potential, and glycolytic activity. When POTS cells were treated with choline, transporter was up-regulated, and uptake of choline increased, offering an option for patient treatment. The characteristics of the POTS fibroblasts described here represent a first model of choline and CTL1/SLC44A1 deficiency, in which choline transport, membrane homeostasis, and mitochondrial function are impaired.

  2. Artificial Skin – Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

    Science.gov (United States)

    Reimers, Kerstin; Kuhbier, Joern W.; Schäfer-Nolte, Franziska; Allmeling, Christina; Kasper, Cornelia; Vogt, Peter M.

    2011-01-01

    Background In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross- sections were stained in Haematoxylin & Eosin (H&E) for microscopic analyses. Conclusion/Significance Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration. PMID:21814557

  3. Artificial skin--culturing of different skin cell lines for generating an artificial skin substitute on cross-weaved spider silk fibres.

    Directory of Open Access Journals (Sweden)

    Hanna Wendt

    Full Text Available BACKGROUND: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. METHODOLOGY/PRINCIPAL FINDINGS: Native spider dragline silk, harvested directly out of Nephila spp spiders, was woven on steel frames. Constructs were sterilized and seeded with fibroblasts. After two weeks of cultivating single fibroblasts, keratinocytes were added to generate a bilayered skin model, consisting of dermis and epidermis equivalents. For the next three weeks, constructs in co-culture were lifted on an originally designed setup for air/liquid interface cultivation. After the culturing period, constructs were embedded in paraffin with an especially developed program for spidersilk to avoid supercontraction. Paraffin cross-sections were stained in Haematoxylin & Eosin (H&E for microscopic analyses. CONCLUSION/SIGNIFICANCE: Native spider dragline silk woven on steel frames provides a suitable matrix for 3 dimensional skin cell culturing. Both fibroblasts and keratinocytes cell lines adhere to the spider silk fibres and proliferate. Guided by the spider silk fibres, they sprout into the meshes and reach confluence in at most one week. A well-balanced, bilayered cocultivation in two continuously separated strata can be achieved by serum reduction, changing the medium conditions and the cultivation period at the air/liquid interphase. Therefore spider silk appears to be a promising biomaterial for the enhancement of skin regeneration.

  4. Mitochondrial ribosomal protein S18-2 evokes chromosomal instability and transforms primary rat skin fibroblasts

    KAUST Repository

    Kashuba, Elena

    2015-05-12

    We have shown earlier that overexpression of the human mitochondrial ribosomal protein MRPS18-2 (S18-2) led to immortalization of primary rat embryonic fibroblasts. The derived cells expressed the embryonic stem cell markers, and cellular pathways that control cell proliferation, oxidative phosphorylation, cellular respiration, and other redox reactions were activated in the immortalized cells. Here we report that, upon overexpression of S18-2 protein, primary rat skin fibroblasts underwent cell transformation. Cells passed more than 300 population doublings, and two out of three tested clones gave rise to tumors in experimental animals. Transformed cells showed anchorage-independent growth and loss of contact inhibition; they expressed epithelial markers, such as E-cadherin and β-catenin. Transformed cells showed increased telomerase activity, disturbance of the cell cycle, and chromosomal instability. Taken together, our data suggest that S18-2 is a newly identified oncoprotein that may be involved in cancerogenesis.

  5. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast.

    Science.gov (United States)

    Seol, Ja Young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin's elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity.

  6. Protective influence of hyaluronic acid on focal adhesion kinase activity in human skin fibroblasts exposed to ethanol

    Directory of Open Access Journals (Sweden)

    Donejko M

    2017-03-01

    Full Text Available Magdalena Donejko,1 Edyta Rysiak,2 Elżbieta Galicka,1 Robert Terlikowski,3 Edyta Katarzyna Głażewska,1 Andrzej Przylipiak1 1Department of Esthetic Medicine, 2Department of Medicinal Chemistry, Faculty of Pharmacy, 3Department of Health Restoration, Medical University of Białystok, Białystok, Poland Aim: The aim of this study was to evaluate the effect of ethanol and hyaluronic acid (HA on cell survival and apoptosis in cultured human skin fibroblasts. Regarding the mechanism of ethanol action on human skin fibroblasts, we investigated cell viability and apoptosis, expression of focal adhesion kinase (FAK, and the influence of HA on those processes. Materials and methods: Studies were conducted in confluent human skin fibroblast cultures that were treated with 25 mM, 50 mM, and 100 mM ethanol or with ethanol and 500 µg/mL HA. Cell viability was examined using methyl thiazolyl tetrazolium (MTT assay and NC-300 Nucleo-Counter. Imaging of the cells using a fluorescence microscope Pathway 855 was performed to measure FAK expression. Results: Depending on the dosage, ethanol decreased cell viability and activated the process of apoptosis in human skin fibroblasts. HA prevented the negative influence of ethanol on cell viability and prevented apoptosis. The analysis of fluorescence imaging using BD Pathway 855 High-Content Bioimager showed the inhibition of FAK migration to the cell nucleus, depending on the increasing concentration of ethanol. Conclusion: This study proves that downregulation of signaling pathway of FAK is involved in ethanol-induced apoptosis in human skin fibroblasts. The work also indicates a protective influence of HA on FAK activity in human skin fibroblasts exposed to ethanol. Keywords: apoptosis, skin fibroblast, focal adhesion kinase, hyaluronic acid, ethanol

  7. Genome-Wide Expression Analysis in Fibroblast Cell Lines from Probands with Pallister Killian Syndrome

    OpenAIRE

    Kaur, Maninder; Izumi, Kosuke; Wilkens, Alisha B.; Chatfield, Kathryn C.; Spinner, Nancy B.; Conlin, Laura K.; Zhang, Zhe; Krantz, Ian D.

    2014-01-01

    Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic...

  8. Characterisation of the kynurenine pathway in skin-derived fibroblasts and keratinocytes.

    Science.gov (United States)

    Sheipouri, Diba; Grant, Ross; Bustamante, Sonia; Lovejoy, David; Guillemin, Gilles J; Braidy, Nady

    2015-06-01

    Acute UVB exposure triggers inflammation leading to the induction of indoleamine 2,3 dioxygenase (IDO1), one of the first enzymes in the kynurenine pathway (KP) for tryptophan degradation. However, limited studies have been undertaken to determine the catabolism of tryptophan within the skin. The aim of this study was two fold: (1) to establish if the administration of the proinflammatory cytokine interferon-gamma (IFN-γ) and/or UVB radiation elicits differential KP expression patterns in human fibroblast and keratinocytes; and (2) to evaluate the effect of KP metabolites on intracellular nicotinamide adenine dinucleotide (NAD(+) ) levels, and cell viability. Primary cultures of human fibroblasts and keratinocytes were used to examine expression of the KP at the mRNA level using qPCR, and at the protein level using immunocytochemistry. Cellular responses to KP metabolites were assessed by examining extracellular lactate dehydrogenase (LDH) activity and intracellular NAD(+) levels. Major downstream KP metabolites were analyzed using GC/MS and HPLC. Our data shows that the KP is fully expressed both in human fibroblasts and keratinocytes. Exposure to UVB radiation and/or IFN-γ causes significant changes in the expression pattern of downstream KP metabolites and enzymes. Exposure to various concentrations of KP metabolites showed marked differences in cell viability and intracellular NAD(+) production, providing support for involvement of the KP in the de novo synthesis of NAD(+) in the skin. This new information will have a significant impact on our understanding of the pathogenesis of UV related skin damage and the diagnosis of KP related disease states.

  9. Microporous dermal-mimetic electrospun scaffolds pre-seeded with fibroblasts promote tissue regeneration in full-thickness skin wounds.

    Directory of Open Access Journals (Sweden)

    Paul P Bonvallet

    Full Text Available Electrospun scaffolds serve as promising substrates for tissue repair due to their nanofibrous architecture and amenability to tailoring of chemical composition. In this study, the regenerative potential of a microporous electrospun scaffold pre-seeded with dermal fibroblasts was evaluated. Previously we reported that a 70% collagen I and 30% poly(Ɛ-caprolactone electrospun scaffold (70:30 col/PCL containing 160 μm diameter pores had favorable mechanical properties, supported fibroblast infiltration and subsequent cell-mediated deposition of extracellular matrix (ECM, and promoted more rapid and effective in vivo skin regeneration when compared to scaffolds lacking micropores. In the current study we tested the hypothesis that the efficacy of the 70:30 col/PCL microporous scaffolds could be further enhanced by seeding scaffolds with dermal fibroblasts prior to implantation into skin wounds. To address this hypothesis, a Fischer 344 (F344 rat syngeneic model was employed. In vitro studies showed that dermal fibroblasts isolated from F344 rat skin were able to adhere and proliferate on 70:30 col/PCL microporous scaffolds, and the cells also filled the 160 μm pores with native ECM proteins such as collagen I and fibronectin. Additionally, scaffolds seeded with F344 fibroblasts exhibited a low rate of contraction (~14% over a 21 day time frame. To assess regenerative potential, scaffolds with or without seeded F344 dermal fibroblasts were implanted into full thickness, critical size defects created in F344 hosts. Specifically, we compared: microporous scaffolds containing fibroblasts seeded for 4 days; scaffolds containing fibroblasts seeded for only 1 day; acellular microporous scaffolds; and a sham wound (no scaffold. Scaffolds containing fibroblasts seeded for 4 days had the best response of all treatment groups with respect to accelerated wound healing, a more normal-appearing dermal matrix structure, and hair follicle regeneration

  10. A New Skin Tensiometer Device: Computational Analyses To Understand Biodynamic Excisional Skin Tension Lines.

    Science.gov (United States)

    Paul, Sharad P; Matulich, Justin; Charlton, Nick

    2016-07-25

    One of the problems in planning cutaneous surgery is that human skin is anisotropic, or directionally dependent. Indeed, skin tension varies between individuals and at different body sites. Many a surgeon has tried to design different devices to measure skin tension to help plan excisional surgery, or to understand wound healing. However, many of the devices have been beset with problems due to many confounding variables - differences in technical ability, material (sutures) used and variability between different users. We describe the development of a new skin tensiometer that overcomes many historical technical issues. A new skin tension measuring device is presented here. It was designed to be less user-dependent, more reliable and usable on different bodily sites. The design and computational optimizations are discussed. Our skin tensiometer has helped understand the differences between incisional and excisional skin lines. Langer, who pioneered the concept of skin tension lines, created incisional lines that differ from lines caused by forces that need to be overcome when large wounds are closed surgically (excisional tension). The use of this innovative device has led to understanding of skin biomechanics and best excisional skin tension (BEST) lines.

  11. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    Science.gov (United States)

    Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin’s elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the media of Human Dermal Fibroblast (NHDF-c) to test anti-aging effects. First, it was identified that growth rate of skin fibroblast was increased. Furthermore, the Hanwoo bone infusion increased a 50% of fibroblast collagen synthesis. Also, suppression of skin fibroblast aging was confirmed by treatment Hanwoo bone infusion. In conclusion, this study demonstrates the effects of infusion made from Hanwoo leg bone, foot and tail on anti-aging, wrinkle inhibiting and skin fibroblast elasticity maintaining. Therefore, this study identified that traditional infusion has effects that are good for skin elasticity. PMID:27194933

  12. Inductive role of fibroblastic cell lines in development of the mouse thymus anlage in organ culture.

    Science.gov (United States)

    Itoi, M; Amagai, T

    1998-01-10

    Previously, we have shown that embryonic day 12 thymus anlage cultured alone cannot develop into the mature organ but degenerates. In the present study, we investigated the cause of this insufficient organogenesis of embryonic day 12 thymus anlage in organ culture. We cocultured embryonic day 12 thymus anlages with various cell lines as pellets formed by centrifugation. In coculture with fibroblastic cell lines, but not with thymic epithelial cell lines, embryonic day 12 thymus anlages developed to support full T cell differentiation, and expressed mature stromal cell markers, Ia and Kb. By pellet culture of thymus anlages and fibroblastic cell lines transfected with a beta-galactosidase expression vector, we analyzed the distribution of added fibroblastic cells in pellets. The added fibroblastic cells constituted neither thymic capsule nor septa but disappeared after about 2 weeks in culture. Moreover, immunohistochemical studies indicated that added fibroblastic cells were adjacent to mesenchymal cells of thymus anlage. Our results strongly suggest that added fibroblastic cells support the development of the thymus anlage through interaction with its mesenchymal cells.

  13. L-Lactate Protects Skin Fibroblasts against Aging-Associated Mitochondrial Dysfunction via Mitohormesis

    Directory of Open Access Journals (Sweden)

    Jaroslav Zelenka

    2015-01-01

    Full Text Available A moderate elevation of reactive oxygen species (ROS production and a mild inhibition of mitochondrial respiratory chain have been associated with a health promotion and a lifespan extension in several animal models of aging. Here, we tested whether this phenomenon called mitohormesis could be mediated by L-lactate. The treatment with 5 mM L-lactate significantly increased H2O2 production and slightly inhibited the respiration in cultured skin fibroblasts and in isolated mitochondria. The L-lactate exposure was associated with oxidation of intracellular glutathione, phosphorylation of 5′AMP-activated protein kinase (AMPK, and induction of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α transcription. A replicative aging of fibroblasts (L0 with a constant (LC, or intermittent 5 mM L-lactate (LI in media showed that the high-passage LI fibroblasts have higher respiration, lower H2O2 release, and lower secretion of L-lactate compared to L0 and LC. This protection against mitochondrial dysfunction in LI cells was associated with lower activity of mechanistic target of rapamycin complex 1 (mTORC1, less signs of cellular senescence, and increased autophagy compared to L0 and LC. In conclusion, we demonstrated that intermittent but not constant exposure to L-lactate triggers mitohormesis, prevents aging-associated mitochondrial dysfunction, and improves other markers of aging.

  14. MORPHOFUNCTIONAL CHARACTERISTICS OF FIBROBLASTS (MCCOY CELL LINE CULTURED WITH MAGNESIUM PREPARATIONS

    Directory of Open Access Journals (Sweden)

    L. V. Didenko

    2015-01-01

    Full Text Available Aim. To study the effect of magnesium orotate, magnesium/pyridoxine combination and magnesium sulfate on fibroblast morphofunctional characteristics in cell culture of fibroblasts (McCoy line.Material and methods. The study of fibroblasts (McCoy line with the addition of magnesium-containing preparations (magnesium orotate, magnesium/pyridoxine combination, magnesium sulphate to the culture medium was performed using scanning and transmission electron microscopy.Results. When adding into the media magnesium orotate or magnesium/pyridoxine combination, significant changes in morphofunctional state of fibroblasts were noted, which were absent when adding magnesium sulfate. At that fibroblasts significantly promoted synthetic and secretory activity. This was expressed in the formation of amorphous and fibrous components in well-formed cell layers.Conclusion. Stimulation by magnesium ions of the proliferative activity of fibroblasts is shown in the morphological analysis of the effect of magnesium orotate or magnesium in combination with pyridoxine and magnesium sulfate on morphological and functional organization of fibroblasts. Revealed the predominant influence of magnesium orotate, and a less pronounced effect of magnesium in combination with pyridoxine on biosynthetic processes in the cells, including the synthesis of amorphous and fibrous components (protocollagen and elastin of the extracellular matrix founded.

  15. Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

    Science.gov (United States)

    Fleischmajer, R; MacDonald, E D; Contard, P; Perlish, J S

    1993-09-01

    Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

  16. Gallic acid regulates skin photoaging in UVB-exposed fibroblast and hairless mice.

    Science.gov (United States)

    Hwang, Eunson; Park, Sang-Yong; Lee, Hyun Ji; Lee, Tae Youp; Sun, Zheng-Wang; Yi, Tae Hoo

    2014-12-01

    Ultraviolet (UV) radiation is the primary factor in skin photoaging, which is characterized by wrinkle formation, dryness, and thickening. The mechanisms underlying skin photoaging are closely associated with degradation of collagen via upregulation of matrix metalloproteinase (MMP) activity, which is induced by reactive oxygen species (ROS) production. Gallic acid (GA), a phenolic compound, possesses a variety of biological activities including antioxidant and antiinflammatory activities. We investigated the protective effects of GA against photoaging caused by UVB irradiation using normal human dermal fibroblasts (NHDFs) in vitro and hairless mice in vivo. The production levels of ROS, interlukin-6, and MMP-1 were significantly suppressed, and type I procollagen expression was stimulated in UVB-irradiated and GA-treated NHDFs. GA treatment inhibited the activity of transcription factor activation protein 1. The effects of GA following topical application and dietary administration were examined by measuring wrinkle formation, histological modification, protein expression, and physiological changes such as stratum corneum hydration, transepidermal water loss, and erythema index. We found that GA decreased dryness, skin thickness, and wrinkle formation via negative modulation of MMP-1 secretion and positive regulation of elastin, type I procollagen, and transforming growth factor-β1. Our data indicate that GA is a potential candidate for the prevention of UVB-induced premature skin aging.

  17. In vitro study of RRS HA injectable mesotherapy/biorevitalization product on human skin fibroblasts and its clinical utilization

    Directory of Open Access Journals (Sweden)

    Deglesne PA

    2016-02-01

    Full Text Available Pierre-Antoine Deglesne,* Rodrigo Arroyo,* Evgeniya Ranneva, Philippe Deprez Research and Development, SKIN TECH PHARMA GROUP, Castelló d'Empúries, Spain  *These authors contributed equally to this work Abstract: Mesotherapy/biorevitalization with hyaluronic acid (HA is a treatment approach currently used for skin rejuvenation. Various products with a wide range of polycomponent formulations are available on the market. Most of these formulations contain noncross-linked HA in combination with a biorevitalization cocktail, formed by various amounts of vitamins, minerals, amino acids, nucleotides, coenzymes, and antioxidants. Although ingredients are very similar among the different products, in vitro and clinical effects may vary substantially. There is a real need for better characterization of these products in terms of their action on human skin or in vitro skin models. In this study, we analyzed the effect of the RRS® (Repairs, Refills, Stimulates HA injectable medical device on human skin fibroblasts in vitro. Skin fibroblast viability and its capacity to induce the production of key extracellular matrix were evaluated in the presence of different concentrations of RRS HA injectable. Viability was evaluated through colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, and key extracellular matrix genes, type I collagen and elastin, were quantified by quantitative polymerase chain reaction. Results demonstrated that RRS HA injectable could promote human skin fibroblast viability (+15% and increase fibroblast gene expression of type I collagen and elastin by 9.7-fold and 14-fold in vitro, respectively. These results demonstrate that mesotherapy/biorevitalization products can, at least in vitro, effectively modulate human skin fibroblasts.Keywords: mesotherapy, medical device, RRS, collagen, elastin, extracellular matrix

  18. Zinc and propolis reduces cytotoxicity and proliferation in skin fibroblast cell culture: total polyphenol content and antioxidant capacity of propolis.

    Science.gov (United States)

    Tyszka-Czochara, Małgorzata; Paśko, Paweł; Reczyński, Witold; Szlósarczyk, Marek; Bystrowska, Beata; Opoka, Włodzimierz

    2014-07-01

    It has been demonstrated that zinc exerts its beneficial influence on skin fibroblasts. Propolis, a complex mixture of plant-derived and bees' products, was reported to stimulate cicatrization processes in skin and prevent infections. The aim of this study was to find out how zinc and propolis influence human skin fibroblasts in cell culture and to compare the effect of individual compounds to the effect of a mixture of zinc and propolis. In this study, zinc, as zinc aspartate, at a concentration of 16 μM, increased human fibroblasts proliferation in cell culture, whereas propolis at a concentration of 0.01% (w/v) revealed antiproliferative and cytotoxic action followed by mild cell necrosis. In culture, zinc was effectively transported into fibroblasts, and propolis inhibited the amount of zinc incorporated into the cells. An addition of propolis to the medium caused a decrease in the Zn(II) amount incorporated into fibroblasts. The obtained results also indicate an appreciable antioxidant property of propolis and revealed its potential as a supplement when applied at doses lower than 0.01% (w/v). In conclusion, the present study showed that zinc had a protective effect on human cultured fibroblasts' viability, although propolis revealed its antiproliferative action and caused mild necrosis.

  19. Resveratrol Prevents High Fluence Red Light-Emitting Diode Reactive Oxygen Species-Mediated Photoinhibition of Human Skin Fibroblast Migration.

    Directory of Open Access Journals (Sweden)

    Andrew Mamalis

    Full Text Available Skin fibrosis is a significant medical problem that leads to a functional, aesthetic, and psychosocial impact on quality-of-life. Light-emitting diode-generated 633-nm red light (LED-RL is part of the visible light spectrum that is not known to cause DNA damage and is considered a safe, non-invasive, inexpensive, and portable potential alternative to ultraviolet phototherapy that may change the treatment paradigm of fibrotic skin disease.The goal of our study was to investigate the how reactive oxygen species (ROS free radicals generated by high fluence LED-RL inhibit the migration of skin fibroblasts, the main cell type involved in skin fibrosis. Fibroblast migration speed is increased in skin fibrosis, and we studied cellular migration speed of cultured human skin fibroblasts as a surrogate measure of high fluence LED-RL effect on fibroblast function. To ascertain the inhibitory role of LED-RL generated ROS on migration speed, we hypothesized that resveratrol, a potent antioxidant, could prevent the photoinhibitory effects of high fluence LED-RL on fibroblast migration speed.High fluence LED-RL generated ROS were measured by flow cytometry analysis using dihydrorhodamine (DHR. For purposes of comparison, we assessed the effects of ROS generated by hydrogen peroxide (H2O2 on fibroblast migration speed and the ability of resveratrol, a well known antioxidant, to prevent LED-RL and H2O2 generated ROS-associated changes in fibroblast migration speed. To determine whether resveratrol could prevent the high fluence LED-RL ROS-mediated photoinhibition of human skin fibroblast migration, treated cells were incubated with resveratrol at concentrations of 0.0001% and 0.001% for 24 hours, irradiated with high fluences LED-RL of 480, 640, and 800 J/cm2.High fluence LED-RL increases intracellular fibroblast ROS and decreases fibroblast migration speed. LED-RL at 480, 640 and 800 J/cm2 increased ROS levels to 132.8%, 151.0%, and 158.4% relative to matched

  20. Superficial Dermal Fibroblasts Enhance Basement Membrane and Epidermal Barrier Formation in Tissue-Engineered Skin: Implications for Treatment of Skin Basement Membrane Disorders

    OpenAIRE

    2013-01-01

    Basement membrane is a highly specialized structure that binds the dermis and the epidermis of the skin, and is mainly composed of laminins, nidogen, collagen types IV and VII, and the proteoglycans, collagen type XVIII and perlecan, all of which play critical roles in the function and resilience of skin. Both dermal fibroblasts and epidermal keratinocytes contribute to the development of the basement membrane, and in turn the basement membrane and underlying dermis influence the development ...

  1. Endothelin receptor antagonists: effects on extracellular matrix synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients

    Directory of Open Access Journals (Sweden)

    B. Villaggio

    2012-12-01

    Full Text Available Endothelin-1 (ET-1 seems to enhance the pro-fibrotic protein synthesis by skin fibroblasts and its effects are mediated by endothelin-A and B (ETA and ETB receptors. This study aimed to investigate the effects of ETA and ETB receptor antagonists (ETARA-sitaxentan and ETA/BRA-bosentan on type I collagen (COL-1, fibronectin (FN and fibrillin-1 (FBL-1 synthesis in primary cultures of skin fibroblasts from systemic sclerosis patients. Primary cultures of fibroblasts were obtained from skin biopsies of 6 female systemic sclerosis patients and were treated with ET-1 (100 nM for 24 and 48 hrs with or without pre-treatment (1 hr with ETARA (2 μM or ETA/BRA (10 μM. Primary culture of human scleroderma skin fibroblasts not treated with ET-1 or ET receptor antagonists (ETARA and ETA/BRA were used as controls. COL-1, FN and FBL-1 synthesis was evaluated by immunocytochemistry and Western blot analysis. Immunocytochemistry and Western blot analysis showed that ET-1 significantly increased COL-1 and FN synthesis at 24 and 48 hrs and FBL-1 synthesis at 48 hrs vs untreated cells. ETARA significantly contrasted the ET-1-mediated increase in COL-1 and FN at 24 hrs as well as COL-1 and FBL-1 at 48 hrs, but not FN synthesis vs ET-1-treated fibroblasts. Conversely, ETA/BRA significantly antagonized the ET-1-mediated overproduction of COL-1 and FN both at 24 and 48 hrs and the FBL-1 synthesis at 48 hrs vs ET-1-treated cells. The single ETARA treatment seems to contrast significantly the increase in COL-1 synthesis, whereas the dual ETA/BRA treatment seems active in significantly antagonizing both COL-1 and FN overproduction induced by ET-1. In conclusion, ET-1 antagonism might have positive effects in contrasting the profibrotic activity of systemic sclerosis skin fibroblasts.

  2. Effects of bosentan on collagen type I synthesis on in vitro culture of scleroderma skin fibroblasts

    Directory of Open Access Journals (Sweden)

    S. Soldano

    2011-01-01

    Full Text Available The present study evaluated the effects of a non-selective endothelin (ETA/B receptors antagonist, on collagen type I (COLI synthesis on in vitro culture of scleroderma (SSc skin fibroblasts (Fb. Fb were obtained from skin biopsies of 6 female SSc patients (mean age 64. 1±6 years, after informed consent and Ethical Committee Approval. Cells were treated with endothelin-I [ET-I, 100nM] for 24 and 48 hrs, pre-treated for I hr with ETA/B receptors antagonist [10nM] alone or followed by ET-I for 24 and 48 hrs. Untreated Fb were used as controls. Immunocytochemistry and western blot analysis were performed to evaluate COLI synthesis. ET-I increased COLI synthesis both at 24 and 48 hrs when compared to controls. ETA/B receptor antagonost blocks the increased COLI synthesis ET-I-mediated both at 24 and 48 hrs vs. ET-I. Results showed that ET-I receptors blockage by ETA/B receptors antagonist might prevent the excessive synthesis of COLI, supporting its positive action in the management of skin fibrosis.

  3. Content of Androgen Receptor in Cultured Genital Skin Fibroblast From Different Ages of Chinese Normal Men

    Institute of Scientific and Technical Information of China (English)

    卢建; 何立敏; 张金山; 杨震; 周云

    1995-01-01

    A ratpid, simple, reliable method is described for assaying androgen receptor (AR) in dispersed, whole, cultured human genital skin fibroblasts (GSF) with a synthetic androgen, 3H-methyltrienolone (3H-R1881). Receptors for androgen in GSF exhiblt high affinity (Kd=3.0±0.1 nmol/L), low binding capacity and androgen specificity. The content of AR in cultured GSF from 40 normal men varying in age from 1.5—60 years u:as also investigated by this assay. Scatchard analysis and slngle plot revealed the presence of 4.500-8500 binding sites per cell, mean number of AR in GSF of these men is 6288±1082 binding sites/cell. No significant difference was observed in the content of AR in different age groups. This result showed that the content of AR in these ceils did not change with age.

  4. Induction of micronuclei in vitro by organochlorine compounds in beluga whale skin fibroblasts.

    Science.gov (United States)

    Gauthier, J M; Dubeau, H; Rassart, E

    1999-02-02

    Beluga whales (Delphinapterus leucas) inhabiting the St. Lawrence estuary are highly contaminated with environmental pollutants and have a high incidence of cancer. Environmental contaminants may be partly responsible for the high cancer incidence observed in this population. DNA damage plays an important role in the development of cancer. The micronuclei (MN) assay was used to test the genotoxic potential of organochlorine (OC) pesticides with and without external metabolic factor in skin fibroblasts of an Arctic beluga whale. Toxaphene, chlordane and p,p'-DDT induced significant (pSt. Lawrence beluga whales. However, because of the known genotoxicity of toxaphene and the long lifespan of beluga whales, it cannot be excluded that toxaphene may pose a long-term genetic hazard to the more contaminated whales of this population.

  5. Estrogen prevents oxidative damage to the mitochondria in Friedreich's ataxia skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Timothy E Richardson

    Full Text Available Estrogen and estrogen-related compounds have been shown to have very potent cytoprotective properties in a wide range of disease models, including an in vitro model of Friedreich's ataxia (FRDA. This study describes a potential estrogen receptor (ER-independent mechanism by which estrogens act to protect human FRDA skin fibroblasts from a BSO-induced oxidative insult resulting from inhibition of de novo glutathione (GSH synthesis. We demonstrate that phenolic estrogens, independent of any known ER, are able to prevent lipid peroxidation and mitochondrial membrane potential (ΔΨm collapse, maintain ATP at near control levels, increase oxidative phosphorylation and maintain activity of aconitase. Estrogens did not, however, prevent BSO from depleting GSH or induce an increased expression level of GSH. The cytoprotective effects of estrogen appear to be due to a direct overall reduction in oxidative damage to the mitochondria, enabling the FRDA fibroblast mitochondria to generate sufficient ATP for energy requirements and better survive oxidative stress. These data support the hypothesis that phenol ring containing estrogens are possible candidate drugs for the delay and/or prevention of FRDA symptoms.

  6. Cytotoxic and Oxidative Stress Caused by Cadmium and Lead on Human Skin Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Ali Beman Zaree Mahmodabady

    2006-01-01

    Full Text Available Introduction: Heavy metals are important occupational andenvironmental pollutants that cause damage to various organs.Although there is no effective therapy for such a poisoning,metallothionein has been shown to play a key role in thedetoxification of cadmium (Cd. Evidence in the literature suggeststhat superoxide dismutase, glutathione peroxidase, and catalaseconstitute important defense mechanisms against oxygen toxicity inthe cells. The aim of this study was to investigate the effect ofcadmium chloride and Pb-acetate on antioxidant enzymes in thehuman skin fibroblast cells (HF2FF.Material and Methods: The human skin fibroblast (HF2FF cellswere incubated in serum-free medium containing 20 μM CdCl2 for18 hr three times a week. The same exposure to an equimolar doseof Pb-acetate was performed. After each exposure and after threetimes exposure the cells were collected and cell viability, thecontents of superoxide dismutase (SOD, catalase, glutathioneperoxidase (GSH-Px, GSH and malondialdehyde (MDA weremeasured.Results: Cd caused cytotoxicity and inhibition of glutathioneperoxidase (GSH-Px and SOD activity, as well as depletion of thereduced form of glutathione (GSH in the cell. The level of lipidperoxidation (LP was increased, but catalase activity was notsignificantly altered. These defects were increased with repeatedexposures. The same exposure to an equimolar dose of Pb-acetateevoked only inhibition of GSH-Px and SOD. The values of GSH,catalase and LP activity remained unchanged.Conclusion: The inhibition of GSH-Px and SOD may be consideredas an important biomarker of the toxic effect of metals.

  7. Dramatic Increase in Oxidative Stress in Carbon-Irradiated Normal Human Skin Fibroblasts

    Science.gov (United States)

    Laurent, Carine; Leduc, Alexandre; Pottier, Ivannah; Prévost, Virginie; Sichel, François; Lefaix, Jean-Louis

    2013-01-01

    Skin complications were recently reported after carbon-ion (C-ion) radiation therapy. Oxidative stress is considered an important pathway in the appearance of late skin reactions. We evaluated oxidative stress in normal human skin fibroblasts after carbon-ion vs. X-ray irradiation. Survival curves and radiobiological parameters were calculated. DNA damage was quantified, as were lipid peroxidation (LPO), protein carbonylation and antioxidant enzyme activities. Reduced and oxidized glutathione ratios (GSH/GSSG) were determined. Proinflammatory cytokine secretion in culture supernatants was evaluated. The relative biological effectiveness (RBE) of C-ions vs. X-rays was 4.8 at D0 (irradiation dose corresponding to a surviving fraction of 37%). Surviving fraction at 2 Gy (SF2) was 71.8% and 7.6% for X-rays and C-ions, respectively. Compared with X-rays, immediate DNA damage was increased less after C-ions, but a late increase was observed at D10% (irradiation dose corresponding to a surviving fraction of 10%). LPO products and protein carbonyls were only increased 24 hours after C-ions. After X-rays, superoxide dismutase (SOD) activity was strongly increased immediately and on day 14 at D0% (irradiation dose corresponding to a surviving fraction of around 0%), catalase activity was unchanged and glutathione peroxidase (GPx) activity was increased only on day 14. These activities were decreased after C-ions compared with X-rays. GSH/GSSG was unchanged after X-rays but was decreased immediately after C-ion irradiation before an increase from day 7. Secretion of IL-6 was increased at late times after X-ray irradiation. After C-ion irradiation, IL-6 concentration was increased on day 7 but was lower compared with X-rays at later times. C-ion effects on normal human skin fibroblasts seemed to be harmful in comparison with X-rays as they produce late DNA damage, LPO products and protein carbonyls, and as they decrease antioxidant defences. Mechanisms leading to this

  8. Human amniotic fluid derived cells can competently substitute dermal fibroblasts in a tissue-engineered dermo-epidermal skin analog

    NARCIS (Netherlands)

    Hartmann-Fritsch, Fabienne; Hosper, Nynke; Luginbuehl, Joachim; Biedermann, Thomas; Reichmann, Ernst; Meuli, Martin

    2013-01-01

    Human amniotic fluid comprises cells with high differentiation capacity, thus representing a potential cell source for skin tissue engineering. In this experimental study, we investigated the ability of human amniotic fluid derived cells to substitute dermal fibroblasts and support epidermis formati

  9. Altered beta-adrenergic receptor-stimulated cAMP formation in cultured skin fibroblasts from Alzheimer donors.

    Science.gov (United States)

    Huang, H M; Gibson, G E

    1993-07-15

    An alteration in signal transduction systems in Alzheimer's disease would likely be of pathophysiological significance, because these steps are critical to normal brain function. Since dynamic processes are difficult to study in autopsied brain, the current studies utilized cultured skin fibroblasts. The beta-adrenergic-stimulated increase in cAMP was reduced approximately 80% in fibroblasts from Alzheimer's disease compared with age-matched controls. The deficit in Alzheimer fibroblasts in response to various adrenergic agonists paralleled their beta-adrenergic potency, and enhancement of cAMP accumulation by a non-adrenergic agonist, such as prostaglandin E1, was similar in Alzheimer and control fibroblasts. Diminished adenylate cyclase activity did not underlie these abnormalities, since direct stimulation of adenylate cyclase by forskolin elevated cAMP production equally in Alzheimer and control fibroblasts. Cholera toxin equally stimulated cAMP formation in Alzheimer and control fibroblasts. Moreover, cholera toxin partially reduced isoproterenol-induced cAMP deficit in Alzheimer fibroblasts. Pertussis toxin, on the other hand, did not alter the Alzheimer deficits. The results suggest either that the coupling of the GTP-binding protein(s) to the beta-adrenergic receptor is abnormal or that the sensitivity of receptor is altered with Alzheimer's disease. Further, any hypothesis about Alzheimer's disease must explain why a reduced beta-adrenergic-stimulated cAMP formation persists in tissue culture.

  10. 12p microRNA expression in fibroblast cell lines from probands with Pallister-Killian syndrome.

    Science.gov (United States)

    Izumi, Kosuke; Zhang, Zhe; Kaur, Maninder; Krantz, Ian D

    2014-12-01

    Pallister-Killian syndrome is a multisystem sporadic genetic diagnosis characterized by facial dysmorphia, variable developmental delay and intellectual impairment, hypotonia, seizures, diaphragmatic hernia, and other systemic abnormalities. Pallister-Killian syndrome is typically caused by the presence of a supernumerary isochromosome that is always present in a tissue limited mosaic pattern, resulting in tetrasomy 12p due to the two extra copies of 12p. We evaluated the potential contribution of microRNAs located on 12p to the pathogenesis of Pallister-Killian syndrome phenotype. Using skin fibroblast cell lines from 13 probands with Pallister-Killian syndrome and 5 normal matched controls, the expression level of 5 microRNAs located on 12p and their target gene mRNA levels were measured. All measured micro RNAs located on 12p were overexpressed in Pallister-Killian syndrome fibroblasts, although the fold difference of the expression level was lower than copy number differences. Among the five microRNAs, miR-1244 had the highest fold difference. Many of computer-predicted target genes of miR-1244 were downregulated in Pallister-Killian syndrome skin fibroblasts. In particular, expression levels of MEIS2 and UQCRB were significantly decreased in Pallister-Killian syndrome samples, and an inverse linear correlation was seen between the level of miR-1244 and MEIS2 and UQCRB expression levels. Since many of computer-predicted miR-1244 target genes play roles in transcriptional regulation, overexpression of miR-1244 due to tetrasomy 12p may contribute to the pleiotropic phenotype of Pallister-Killian syndrome by modulating its downstream target genes including MEIS2 and UQCRB.

  11. Cultured skin keratinocytes from both normal individuals and basal cell naevus syndrome patients are more resistant to. gamma. -rays and UV light compared with cultured skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Stacey, M.; Thacker, S.; Taylor, A.M.R. (Birmingham Univ. (UK). Cancer Research Campaign Labs.)

    1989-07-01

    Measurement of colony-forming ability following exposure to {gamma}-rays was performed on cultured skin keratinocytes and skin fibroblasts from normal individuals, basal cell naevus syndrome patients (BCNS) and ataxia telangiectasia (A-T) patients. The most striking observation was the radiation resistance of 8/8 keratinocyte strains compared with fibroblasts whether from BCNS patients or normals. The single A-T keratinocyte cell strain showed the same radiosensitivity as A-T fibroblast cell strains. The differential survival of keratinocytes and fibroblasts was also observed following exposure to 254 nm UV light. Survival curves of SV40 immortalized keratinocytes and retinoblasts derived from normal individuals or BCNS patients showed large shoulder regions following exposure to {gamma}-rays or 254 nm UV light. An increased D{sub 37} rather than an increased D{sub o} was therefore the feature of such curves, contrasting with SV40 immortalized A-T keratinocytes or fibroblasts which showed a minimal shoulder effect and an increased D{sub o}. No difference in survival was observed between BCNS and normal cells following exposure to either UV or {gamma}-rays. (author).

  12. Aloin Protects Skin Fibroblasts from Heat Stress-Induced Oxidative Stress Damage by Regulating the Oxidative Defense System.

    Directory of Open Access Journals (Sweden)

    Fu-Wei Liu

    Full Text Available Oxidative stress is commonly involved in the pathogenesis of skin damage induced by environmental factors, such as heat stress. Skin fibroblasts are responsible for the connective tissue regeneration and the skin recovery from injury. Aloin, a bioactive compound in Aloe vera, has been reported to have various pharmacological activities, such as anti-inflammatory effects. The aim of this study was to investigate the protective effect of aloin against heat stress-mediated oxidative stress in human skin fibroblast Hs68 cells. Hs68 cells were first incubated at 43°C for 30 min to mimic heat stress. The study was further examined if aloin has any effect on heat stress-induced oxidative stress. We found that aloin protected Hs68 cells against heat stress-induced damage, as assessed by 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assay. Aloin protected Hs68 cells by regulating reactive oxygen species production and increasing the levels of glutathione, cytosolic and mitochondrial superoxide dismutase. Aloin also prevented the elevation of thiobarbituric acid reactive substances and the reduction of 8-OH-dG induced by heat stress. These results indicated that aloin protected human skin fibroblasts from heat stress-induced oxidative stress damage by regulating the oxidative defense system.

  13. Protective effect against oxygen reactive species and skin fibroblast stimulation of Couroupita guianensis leaf extracts.

    Science.gov (United States)

    Martínez, Ana; Conde, Enma; Moure, Andrés; Domínguez, Herminia; Estévez, Ramón J

    2012-01-01

    In this study, hydroalcoholic leaf extracts of Couroupita guianensis were examined for antioxidant activity, phytochemical and total phenolic composition, stimulation of human skin fibroblast (HSF) proliferation and UV-absorption. The radical scavenging capacity, reducing power and protection against joint oxidation of linoleic acid and β-carotene bleaching oxidation in emulsion were used to evaluate the antioxidant activity. The results of this study strongly indicate in vitro antioxidant activity, which may be due to the presence of a high total phenolic content. In order to identify active principles, the extracts were submitted to fractionation and the compounds isolated were the flavonoids 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (1), 7-hydroxy-5-methoxy-6,8-dimethylflavanone (2) and the phenolic acid 4-hydroxybenzoic acid (3). In addition, a high level of stimulation of HSF proliferation and significant absorption of UV radiation were also observed. The results suggest that the hydroalcoholic leaf extracts of C. guianensis have promising skin care properties.

  14. Replacement of animal-derived collagen matrix by human fibroblast-derived dermal matrix for human skin equivalent products.

    Science.gov (United States)

    El Ghalbzouri, Abdoelwaheb; Commandeur, Suzan; Rietveld, Marion H; Mulder, Aat A; Willemze, Rein

    2009-01-01

    Reconstructed human skin equivalents (HSEs) are representative models of human skin and widely used for research purposes and clinical applications. Traditional methods to generate HSEs are based on the seeding of human keratinocytes onto three-dimensional human fibroblast-populated non-human collagen matrices. Current HSEs have a limited lifespan of approximately 8 weeks, rendering them unsuitable for long-term studies. Here we present a new generation of HSEs being fully composed of human components and which can be cultured up to 20 weeks. This model is generated on a primary human fibroblast-derived dermal matrix. Pro-collagen type I secretion by human fibroblasts stabilized during long-term culture, providing a continuous and functional human dermal matrix. In contrast to rat-tail collagen-based HSEs, the present fibroblast-derived matrix-based HSEs contain more continuity in the number of viable cell layers in long-term cultures. In addition, these new skin models exhibit normal differentiation and proliferation, based on expression of K10/K15, and K16/K17, respectively. Detection of collagen types IV and VII and laminin 332 was confined to the epidermal-dermal junction, as in native skin. The presence of hemidesmosomes and anchoring fibrils was demonstrated by electron microscopy. Finally, we show that the presented HSE contained a higher concentration of the normal moisturizing factor compared to rat-tail collagen-based skin models, providing a further representation of functional normal human skin in vitro. This study, therefore, demonstrates the role of the dermal microenvironment on epidermal regeneration and lifespan in vitro.

  15. Biochemical mechanisms of skin radiation burns inhibition and healing by the volumetric autotransplantation of fibroblasts and of keratinocytes with fibroblasts composition

    Directory of Open Access Journals (Sweden)

    L. V. Altukhova

    2015-09-01

    Full Text Available Mechanisms of influence of volumetric autotransplantation of fibroblasts and of the mixture of fibroblasts and keratinocytes on the development of the local 3rd degree X-ray burn and the radiation skin ulcer in guinea pigs were investigated. We used deepadministration into the irradiation zone on its perimeter of 6 doses, which contained (150–160×103 fibroblasts and (130–140×103 keratinocytes in 100 µl. It is shown that this autotransplantation carried out 1 hour after the irradiation, and then every 24 hours, reduces the area of burn on the 35th day, compared to the control by 63%. Radiation ulcer appears on the 10th day after irradiation and is completely healed on the 25th day. With the same regimen of administration of only fibroblasts containing (200–210×103 cells in 100 µl, these parameters of treatment were equal to 31% on 4th and 35th day, respectively. It is shown that as a result of radiation in the area of burn the level of gene expression of collagen types I and III, elastin, fibronectin, vinculin, decorin, hyaluronansynthases 1, 2, 3, matrix metalloproteinases 1, 2, 3, 7, 9 and hyaluronidase is reduced. Besides, in the burn area the level of gene expression of transforming growth factor α, fibroblast growth factors 1, 2, 8 and anti-inflammatory cytokines – interleukin 10 and transforming growth factor-β1 – is reduced, while the level of gene expression of proinflammatory cytokine (interleykin1β increases. Both types of autotransplantation cause the growth of the expression level of all the structural genes and regulatory proteins of biopolymers and decrease in the expression level of interleukin 1β, which leads to activation of tissue regeneration and healing of the burn wound. Reasonsfor the higher efficiency of autotransplantation using the mixture of fibroblasts and keratinocytes compared to autotransplantation by fibroblasts only are both the larger total number of live cells regularly replacing dead cells in

  16. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    Science.gov (United States)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  17. Deciphering the differential response of two human fibroblast cell lines following Chikungunya virus infection

    Directory of Open Access Journals (Sweden)

    Thon-Hon Vincent G

    2012-09-01

    Full Text Available Abstract Background Chikungunya virus (CHIKV is an arthritogenic member of the Alphavirus genus (family Togaviridae transmitted by Aedes mosquitoes. CHIKV is now known to target non hematopoietic cells such as epithelial, endothelial cells, fibroblasts and to less extent monocytes/macrophages. The type I interferon (IFN response is an early innate immune mechanism that protects cells against viral infection. Cells express different pattern recognition receptors (including TLR7 and RIG-I to sense viruses and to induce production of type I IFNs which in turn will bind to their receptor. This should result in the phosphorylation and translocation of STAT molecules into the nucleus to promote the transcription of IFN-stimulated antiviral genes (ISGs. We herein tested the capacity of CHIKV clinical isolate to infect two different human fibroblast cell lines HS 633T and HT-1080 and we analyzed the resulting type I IFN innate immune response. Methods Indirect immunofluorescence and quantitative RT-PCR were used to test for the susceptibility of both fibroblast cell lines to CHIKV. Results Interestingly, the two fibroblast cell lines HS 633T and HT-1080 were differently susceptible to CHIKV infection and the former producing at least 30-fold higher viral load at 48 h post-infection (PI. We found that the expression of antiviral genes (RIG-I, IFN-β, ISG54 and ISG56 was more robust in the more susceptible cell line HS 633T at 48 h PI. Moreover, CHIKV was shown to similarly interfere with the nuclear translocation of pSTAT1 in both cell lines. Conclusion Critically, CHIKV can control the IFN response by preventing the nuclear translocation of pSTAT1 in both fibroblast cell lines. Counter-intuitively, the relative resistance of HT-1080 cells to CHIKV infection could not be attributed to more robust innate IFN- and ISG-dependent antiviral responses. These cell lines may prove to be valuable models to screen for novel mechanisms mobilized differentially by

  18. Identiifcation of the miniature pig inbred line by skin allograft

    Institute of Scientific and Technical Information of China (English)

    MU Yu-lian; WEI Jing-liang; TANG Fang; YANG Shu-lin; WU Zhi-gu; XIA Ying; SUN Tong-zhu; LIU Lan; FENG Shu-tang; WU Tian-wen; LI Kui; LI Jun-you; HE Wei; GAO Qian; ZHOU Wen-fang

    2015-01-01

    Skin grafting has been used as one of the most reliable tests to determine the genetic stability of laboratory animal such as mice and rats inbred line, but no identiifcation of swine inbred lines by skin grafting has been reported. At present, Wuzhishan miniature pig (WZSP) inbred line has acquired the F24 individuals in China. In order to verify whether WZSP inbred line had been cultivated successful y, al ogeneic skin grafts and related research were performed on F20 individuals of WZSP inbreeding population, compared with a control group of autologous transplantation. We observed the transplant recipients’ wounds, detected peripheral blood-related indicators interleukin-2, 4 and 10, CD4+and CD8+lymphocytes, and conducted hematoxylin-eosin (HE) and Masson’s staining of skin to judge whether the immune rejection reactions occurred within 28 days after transplantation. Chr. 7 genomic heterozygosity of 48 WZSP individuals from F20 to F22 was analyzed by high-density single nucleotide polymorphism (SNP) chips (60 000 SNPs). The result showed that there were no signiifcant differences in graft skin, the plasma interleukin-2, 4, 10, CD4+and CD8+, HE and Masson’s staining results between the al ograft and autograft groups, and no immune rejection occurred on the al ograft group. We found that 11 genes in Chr. 7 of major histocompatibility complex (MHC) I and MHC II were homozygous which conifrmed that immune antibody of the al ograft and autograft groups were highly identical and also provided a theoretical basis to no immune rejection occurred on the al ograft in the inbred WZSP. The result proved that the WZSP inbred line had been cultivated successful y for the ifrst time in the world. The test methods also provide a scientiifc basis for the identiifcation of swine and mammal inbred lines.

  19. Extracellular Matrix Modulates Morphology, Growth, Oxidative Stress Response and Functionality of Human Skin Fibroblasts during Aging In Vitro

    DEFF Research Database (Denmark)

    Jørgensen, Peter; Rattan, Suresh

    2014-01-01

    The Hayflick system of cellular aging and replicative senescence in vitro has been used widely in both basic and applied research in biogerontology. The state of replicative senescence is generally considered to be irreversible, but is modifiable by genetic and environmental manipulations. Some...... recent observations indicate that replicative lifespan, senescence and functionality of cells in vitro can be significantly affected by the quality of the extra cellular matrix (ECM). Following up on those reports, here we show that using the ECM prepared from early passage young cells, partial...... rejuvenation of serially passaged human facial skin fibroblasts was possible in pre-senescent middle-aged cells, but not in fully senescent late passage cells. ECM from young cells improved the appearance, viability, stress tolerance and wound healing ability of skin fibroblasts. Furthermore, young ECM...

  20. Photobiomodulation on the proliferation and collagen synthesis of normal human skin fibroblast cells

    Science.gov (United States)

    Cheng, Lei; Liu, Timon Cheng-Yi; Chi, Jin-Quan; Li, Yan; Jin, Hua

    2006-01-01

    Background and Objective: Cultured normal human skin fibroblast cells (HSFs) were once used to study the mechanism of the effects of low intensity He-Ne laser irradiation (LHNL) on wound healing. The proliferation and collagen synthesis of HFSs were modulated by LHNL in different papers, respectively, and both of them are studied in this paper. Study Design/Materials and Methods: The dosage was studied for the same radiation time 300s. The proliferation and collagen synthesis were measured by 3-[4,5-Dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and the spectrophotometric method for the determination of hydroxyproline, respectively. Results: The dose zones were called dose 1, dose 2 and dose 3 from low dose on so that HSF proliferation was inhibited in dose 1 (16, 24 mJ/cm2), and promoted in dose 2 (298, 503, 597mJ/cm2), and the collagen synthesis was inhibited in dose 2 (401, 526 mJ/cm2), and promoted in dose 3 (714, 926, 1539, 1727mJ/cm2), which supports our biological model of photobiomodulation. It was found there is the linear relationship of the effect with dose with dose in each dose zone. Conclusions: The photobiomodulation on the proliferation and collagen synthesis of HSFs might be linearly dose-dependent in limited dosage with radiation time kept constant, which provides a foundation to discuss photobiomodulation on wound healing.

  1. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Qiao Lu

    Full Text Available Human induced pluripotent stem cells (hiPSCs have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  2. Negligible immunogenicity of induced pluripotent stem cells derived from human skin fibroblasts.

    Science.gov (United States)

    Lu, Qiao; Yu, Meixing; Shen, Chongyang; Chen, Xiaoping; Feng, Ting; Yao, Yongchao; Li, Jinrong; Li, Hong; Tu, Wenwei

    2014-01-01

    Human induced pluripotent stem cells (hiPSCs) have potential applications in cell replacement therapy and regenerative medicine. However, limited information is available regarding the immunologic features of iPSCs. In this study, expression of MHC and T cell co-stimulatory molecules in hiPSCs, and the effects on activation, proliferation and cytokine production in allogeneic human peripheral blood mononuclear cells were examined. We found that no-integrate hiPSCs had no MHC-II and T cell co-stimulatory molecules expressions but had moderate level of MHC-I and HLA-G expressions. In contrast to human skin fibroblasts (HSFs) which significantly induced allogeneic T cell activation and proliferation, hiPSCs failed to induce allogeneic CD45+ lymphocyte and CD8+ T cell activation and proliferation but could induce a low level of allogeneic CD4+ T cell proliferation. Unlike HSFs which induced allogeneic lymphocytes to produce high levels of IFN-γ, TNF-α and IL-17, hiPSCs only induced allogeneic lymphocytes to produce IL-2 and IL-10, and promote IL-10-secreting regulatory T cell (Treg) generation. Our study suggests that the integration-free hiPSCs had low or negligible immunogenicity, which may result from their induction of IL-10-secreting Treg.

  3. Mesenchymal stem cells suppress fibroblast proliferation and reduce skin fibrosis through a TGF-β3-dependent activation.

    Science.gov (United States)

    Wu, Yan; Peng, Yan; Gao, Dongyun; Feng, Changjiang; Yuan, Xiaohuan; Li, Houzhong; Wang, Ying; Yang, Lan; Huang, Sha; Fu, Xiaobing

    2015-03-01

    Recent studies showed that transplantation of mesenchymal stem cells (MSCs) significantly decreased tissue fibrosis; however, little attention has been paid to its efficacy on attenuating skin fibrosis, and the mechanism involved in its effect is poorly understood. In this work, we investigated the effects of MSCs on keloid fibroblasts and extracellular matrix deposition through paracrine actions and whether the antifibrotic properties of MSCs involved transforming growth factor-β (TGF-β)-dependent activation. In vitro experiments showed that conditioned media (CM) from MSCs decreased viability, a-smooth muscle actin expression, and collagen secretion of human keloid fibroblasts. In addition, TGF-β3 secreted by MSCs was expressed at high level under inflammatory environment, and blocking the activity of TGF-β3 apparently antagonized the suppressive activity of MSC CM, which demonstrated that TGF-β3 played a preponderant role in preventing collagen accumulation. In vivo studies showed that MSC CM infusion in a mouse dermal fibrosis model induced a significant decrease in skin fibrosis. Histological examination of tissue sections and immunohistochemical analysis for α-smooth muscle actin revealed that TGF-β3 of CM-mediated therapeutic effects could obviously attenuate matrix production and myofibroblast proliferation and differentiation. These findings suggest that TGF-β3 mediates the attenuating effect of MSCs on both the proliferation and extracellular matrix production of human keloid fibroblasts and decreases skin fibrosis of mouse model, thus providing new understanding and MSC-based therapeutic strategy for cutaneous scar treatment.

  4. Genome-wide expression analysis in fibroblast cell lines from probands with Pallister Killian syndrome.

    Science.gov (United States)

    Kaur, Maninder; Izumi, Kosuke; Wilkens, Alisha B; Chatfield, Kathryn C; Spinner, Nancy B; Conlin, Laura K; Zhang, Zhe; Krantz, Ian D

    2014-01-01

    Pallister Killian syndrome (OMIM: # 601803) is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p). The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated) differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.

  5. Genome-wide expression analysis in fibroblast cell lines from probands with Pallister Killian syndrome.

    Directory of Open Access Journals (Sweden)

    Maninder Kaur

    Full Text Available Pallister Killian syndrome (OMIM: # 601803 is a rare multisystem disorder typically caused by tissue limited mosaic tetrasomy of chromosome 12p (isochromosome 12p. The clinical manifestations of Pallister Killian syndrome are variable with the most common findings including craniofacial dysmorphia, hypotonia, cognitive impairment, hearing loss, skin pigmentary differences and epilepsy. Isochromosome 12p is identified primarily in skin fibroblast cultures and in chorionic villus and amniotic fluid cell samples and may be identified in blood lymphocytes during the neonatal and early childhood period. We performed genomic expression profiling correlated with interphase fluorescent in situ hybridization and single nucleotide polymorphism array quantification of degree of mosaicism in fibroblasts from 17 Caucasian probands with Pallister Killian syndrome and 9 healthy age, gender and ethnicity matched controls. We identified a characteristic profile of 354 (180 up- and 174 down-regulated differentially expressed genes in Pallister Killian syndrome probands and supportive evidence for a Pallister Killian syndrome critical region on 12p13.31. The differentially expressed genes were enriched for developmentally important genes such as homeobox genes. Among the differentially expressed genes, we identified several genes whose misexpression may be associated with the clinical phenotype of Pallister Killian syndrome such as downregulation of ZFPM2, GATA6 and SOX9, and overexpression of IGFBP2.

  6. Skin equivalent tissue-engineered construct: co-cultured fibroblasts/ keratinocytes on 3D matrices of sericin hope cocoons.

    Science.gov (United States)

    Nayak, Sunita; Dey, Sancharika; Kundu, Subhas C

    2013-01-01

    The development of effective and alternative tissue-engineered skin replacements to autografts, allografts and xenografts has became a clinical requirement due to the problems related to source of donor tissue and the perceived risk of disease transmission. In the present study 3D tissue engineered construct of sericin is developed using co-culture of keratinocytes on the upper surface of the fabricated matrices and with fibroblasts on lower surface. Sericin is obtained from "Sericin Hope" silkworm of Bombyx mori mutant and is extracted from cocoons by autoclave. Porous sericin matrices are prepared by freeze dried method using genipin as crosslinker. The matrices are characterized biochemically and biophysically. The cell proliferation and viability of co-cultured fibroblasts and keratinocytes on matrices for at least 28 days are observed by live/dead assay, Alamar blue assay, and by dual fluorescent staining. The growth of the fibroblasts and keratinocytes in co-culture is correlated with the expression level of TGF-β, b-FGF and IL-8 in the cultured supernatants by enzyme-linked immunosorbent assay. The histological analysis further demonstrates a multi-layered stratified epidermal layer of uninhibited keratinocytes in co-cultured constructs. Presence of involucrin, collagen IV and the fibroblast surface protein in immuno-histochemical stained sections of co-cultured matrices indicates the significance of paracrine signaling between keratinocytes and fibroblasts in the expression of extracellular matrix protein for dermal repair. No significant amount of pro inflammatory cytokines (TNF-α, IL-1β and nitric oxide) production are evidenced when macrophages grown on the sericin matrices. The results all together depict the potentiality of sericin 3D matrices as skin equivalent tissue engineered construct in wound repair.

  7. Effect of low intensity laser interaction with human skin fibroblast cells using fiber-optic nano-probes.

    Science.gov (United States)

    Pal, Gopalendu; Dutta, Ashim; Mitra, Kunal; Grace, Michael S; Amat, Albert; Romanczyk, Tara B; Wu, Xingjia; Chakrabarti, Kristi; Anders, Juanita; Gorman, Erik; Waynant, Ronald W; Tata, Darrell B

    2007-03-01

    Over the past forty years, many efforts have been devoted to study low power laser light interactions with biological systems. Some of the investigations were performed in-vitro, on bulk cell populations. Our present work was undertaken to apply specially engineered fiber-optic based nano-probes for the precise delivery of laser light on to a single cell and to observe production of low power laser light induced reactive oxygen species (ROS). A normal human skin fibroblast (NHF) cell line was utilized in this investigation and the cells were irradiated under two different schemes of exposure: (1) an entire NHF cell population within a Petri dish using a fan beam methodology, and (2) through the precise delivery of laser energy on to a single NHF cell using fiber-optic nano-probe. Photobiostimulative studies were conducted through variation of laser intensity, exposure time, and the energy dose of exposure. Laser irradiation induced enhancement in the rate of cell proliferation was observed to be dependent on laser exposure parameters and the method of laser delivery. The total energy dose (fluence) had a greater influence on the enhancement in the rate of cellular proliferation than compared to laser intensity. The enhancement in the growth rate was observed to have a finite life-time of several days after the initial laser exposure. Fluorescent life-time imaging of ROS was performed during the nano-based single cell exposure method. The kinetics of ROS generation was found to depend strongly on the laser fluence and not on the laser intensity.

  8. Culture of Ear Skin Fibroblasts from the Red Fox (Vulpesvulpes) and Analysis of the Karyotype%赤狐耳成纤维细胞的体外培养及核型分析

    Institute of Scientific and Technical Information of China (English)

    鲍加荣; 王世勇; 郑军军; 杨福合

    2015-01-01

    This study is to isolate and culture skin fibroblasts from the ear of fox.It propels the research on fox somatic nuclear transfer and transgenic fox.In this study,fox ear skin fibroblasts were obtained by tissue pieced culture.The primary culture was digested by trypsin at a short time for the subculture.The fibroblasts were observed by the electron microscope,which had a good growth status.Then karyotype analysis was conduc-ted on the fibroblasts,which showed a standard karyotype of fox.The results showed that fox ear skin fibroblasts could be cultivated successfully in vitro by means of tissues culture method.The primary culture could be puri-fied and ear fibroblast cell line be formed,which can be used as donor cell for nuclear transfer and propels the research on transgenetic special animals.%建立赤狐的耳成纤维细胞的分离和培养体系,为狐的核移植,转基因狐的研究奠定基础.采用组织块培养法分离狐耳成纤维细胞的原代细胞,再利用胰酶短暂消化法进行纯化传代培养,观察成纤维细胞的形态,并进行染色体分析.结果表明,组织块培养法可获得成纤维细胞,经过纯化培养,可获得均一、稳定的狐耳成纤维细胞.可以作为核移植的供体细胞,推动特种动物的转基因研究.

  9. Stimulation of skin repair is dependent on fibroblast source and presence of extracellular matrix.

    NARCIS (Netherlands)

    Wang, H.; Pieper, J.S.; Schotel, R.; Blitterswijk, C.A. van; Lamme, E.N.

    2004-01-01

    In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibrobl

  10. Stimulation of Skin Repair Is Dependent on Fibroblast Source and Presence of Extracellular Matrix

    NARCIS (Netherlands)

    Wang, Hong-Jun; Pieper, Jeroen; Schotel, Roka; Blitterswijk, van Clemens A.; Lamme, Evert N.

    2004-01-01

    In this study in vitro and in vivo functions were compared between cultured dermal equivalents produced with human fibroblasts isolated either from papillary dermis or adipose tissue of the same donors. Papillary dermal fibroblasts had a normal spindle cell shape; in contrast, adipose tissue fibrobl

  11. Lipid peroxidation-derived 4-hydroxynonenal-modified proteins accumulate in human facial skin fibroblasts during ageing in vitro.

    Science.gov (United States)

    Jørgensen, Peter; Milkovic, Lidija; Zarkovic, Neven; Waeg, Georg; Rattan, Suresh I S

    2014-02-01

    The reactive aldehyde, 4-hydroxynonenal (HNE), is recognized as a product of lipid peroxidation, which binds to macromolecules, in particular proteins. HNE-modified proteins (HNE-MP) have been shown to accumulate during ageing, generally by using polyclonal antibodies, which increase the possibility of detecting false positives. Therefore, we have used a genuine monoclonal antibody specific for HNE-His adducts of proteins/peptides, which were revealed by immunoblotting method for whole-cell HNE-MP measurements in serially passaged human facial skin fibroblasts undergoing ageing in vitro. There was a significant increase in the levels of HNE-MP in serially passaged cells approaching a near senescent state at high passage level (P-61), as compared with low passage level (P-11) young and middle-aged (P-27) cells. However, if the cells were analyzed soon after re-initiation from the frozen samples with little further passaging, the amount of HNE-MP was low even in relatively high passage level (P-37) cells, which is an indication of selective elimination of cells with high molecular damage during the process of thawing and re-initiation in culture. This pilot study on normal human facial skin fibroblasts shows that HNE-MP detection by monoclonal antibody-based dot blot method can be used as a marker for age-related accumulation of lipid peroxidative molecular damage, and could be useful for testing and monitoring the effects of potential skin care products on ageing parameters.

  12. DNA damage in wounded, hypoxic and acidotic human skin fibroblast cell cultures after low laser irradiation

    Science.gov (United States)

    Hawkins Evans, D.; Mbene, A.; Zungu, I.; Houreld, N.; Abrahamse, H.

    2009-02-01

    Phototherapy has become more popular and widely used in the treatment of a variety of medical conditions. To ensure sound results as evidence of its effectiveness, well designed experiments must be conducted when determining the effect of phototherapy. Cell culture models such as hypoxic, acidotic and wounded cell cultures simulating different disease conditions including ischemic heart disease, diabetes and wound healing were used to determine the effect of laser irradiation on the genetic integrity of the cell. Even though phototherapy has been found to be beneficial in a wide spectrum of conditions, it has been shown to induce DNA damage. However, this damage appears to be repairable. The risk lies in the fact that phototherapy may help the medical condition initially but damage DNA at the same time leaving undetected damage that may result in late onset, more severe, induced medical conditions including cancer. Human skin fibroblasts were cultured and used to induce a wound (by the central scratch model), hypoxic (by incubation in an anaerobic jar, 95% N2 and 5% O2) and acidotic (reducing the pH of the media to 6.7) conditions. Different models were irradiated using a Helium-Neon (632.8 nm) laser with a power density of 2.07 mW/cm2 and a fluence of 5 J/cm2 or 16 J/cm2. The effect of the irradiation was determined using the Comet assay 1 and 24 h after irradiation. In addition, the Comet assay was performed with the addition of formamidopyrimidine glycosylase (FPG) obviating strand brakes in oxidized bases at a high fluence of 16 J/cm2. A significant increase in DNA damage was seen in all three injured models at both 1 and 24 h post-irradiation when compared to the normal un-injured cells. However, when compared to non-irradiated controls the acidotic model showed a significant decrease in DNA damage 24 h after irradiation indicating the possible induction of cellular DNA repair mechanisms. When wounded cells were irradiated with higher fluences of 16 J/cm2

  13. Effects of sulfate deprivation on the production of chondroitin/dermatan sulfate by cultures of skin fibroblasts from normal and diabetic individuals

    Energy Technology Data Exchange (ETDEWEB)

    Silbert, C.K.; Humphries, D.E.; Palmer, M.E.; Silbert, J.E. (Veterans Administration Outpatient Clinic, Boston, MA (USA))

    1991-02-15

    Human skin fibroblast monolayer cultures from two normal men, three Type I diabetic men, and one Type I diabetic woman were incubated with (3H)glucosamine in the presence of diminished concentrations of sulfate. Although total synthesis of (3H)chondroitin/dermatan glycosaminoglycans varied somewhat between cell lines, glycosaminoglycan production was not affected within any line when sulfate levels were decreased from 0.3 mM to 0.06 mM to 0.01 mM to 0 added sulfate. Lowering of sulfate concentrations resulted in diminished sulfation of chondroitin/dermatan in a progressive manner, so that overall sulfation dropped to as low as 19% for one of the lines. Sulfation of chondroitin to form chondroitin 4-sulfate and chondroitin 6-sulfate was progressively and equally affected by decreasing the sulfate concentration in the culture medium. However, sulfation to form dermatan sulfate was preserved to a greater degree, so that the relative proportion of dermatan sulfate to chondroitin sulfate increased. Essentially all the nonsulfated residues were susceptible to chondroitin AC lyase, indicating that little epimerization of glucuronic acid residues to iduronic acid had occurred in the absence of sulfation. These results confirm the previously described dependency of glucuronic/iduronic epimerization on sulfation, and indicate that sulfation of the iduronic acid-containing disaccharide residues of dermatan can take place with sulfate concentrations lower than those needed for 6-sulfation and 4-sulfation of the glucuronic acid-containing disaccharide residues of chondroitin. There were considerable differences among the six fibroblast lines in susceptibility to low sulfate medium and in the proportion of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate. However, there was no pattern of differences between normals and diabetics.

  14. Exposure to transforming growth factor-β1 after basic fibroblast growth factor promotes the fibroblastic differentiation of human periodontal ligament stem/progenitor cell lines.

    Science.gov (United States)

    Kono, Kiyomi; Maeda, Hidefumi; Fujii, Shinsuke; Tomokiyo, Atsushi; Yamamoto, Naohide; Wada, Naohisa; Monnouchi, Satoshi; Teramatsu, Yoko; Hamano, Sayuri; Koori, Katsuaki; Akamine, Akifumi

    2013-05-01

    Basic fibroblast growth factor (bFGF) is a cytokine that promotes the regeneration of the periodontium, the specialized tissues supporting the teeth. bFGF, does not, however, induce the synthesis of smooth muscle actin alpha 2 (ACTA2), type I collagen (COL1), or COL3, which are principal molecules in periodontal ligament (PDL) tissue, a component of the periodontium. We have suggested the feasibility of using transforming growth factor-β1 (TGFβ1) to induce fibroblastic differentiation of PDL stem/progenitor cells (PDLSCs). Here, we investigated the effect of the subsequent application of TGFβ1 after bFGF (bFGF/TGFβ1) on the differentiation of PDLSCs into fibroblastic cells. We first confirmed the expression of bFGF and TGFβ1 in rat PDL tissue and primary human PDL cells. Receptors for both bFGF and TGFβ1 were expressed in the human PDLSC lines 1-11 and 1-17. Exposure to bFGF for 2 days promoted vascular endothelial growth factor gene and protein expression in both cell lines and down-regulated the expression of ACTA2, COL1, and COL3 mRNA in both cell lines and the gene fibrillin 1 (FBN1) in cell line 1-11 alone. Furthermore, bFGF stimulated cell proliferation of these cell lines and significantly increased the number of cells in phase G2/M in the cell lines. Exposure to TGFβ1 for 2 days induced gene expression of ACTA2 and COL1 in both cell lines and FBN1 in cell line 1-11 alone. BFGF/TGFβ1 treatment significantly up-regulated ACTA2, COL1, and FBN1 expression as compared with the group treated with bFGF alone or the untreated control. This method might thus be useful for accelerating the generation and regeneration of functional periodontium.

  15. Characterization of a novel fibroblast-like cell line from rainbow trout and responses to sublethal anoxia

    DEFF Research Database (Denmark)

    Ossum, Carlo Gunnar; Hoffmann, Else Kay; Vijayan, M.M.;

    2004-01-01

    A novel fibroblast-like cell line RTHDF was established from hypodermal connective tissue of rainbow trout Oncorhynchus mykiss and telomerase activity was demonstrated early and late in cell line development. When RTHDF cells were exposed to bioenergetic stress, i.e. anoxia, activation...... suggests that RTHDF can be useful in studying biochemical responses of teleost cells to environmental stress....

  16. Molecular profiling reveals diversity of stress signal transduction cascades in highly penetrant Alzheimer's disease human skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Graziella Mendonsa

    Full Text Available The serious and growing impact of the neurodegenerative disorder Alzheimer's disease (AD as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer's disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival

  17. A spectroscopic study on the effect of ultra-violet solar radiation in Antarctica on the human skin fibroblast cells

    Directory of Open Access Journals (Sweden)

    Tatsuyuki Yamamoto

    2013-11-01

    Full Text Available A study on the effect of the solar ultra-violet radiation on the human skin fibroblast cells revealed that the production of matrix metalloproteinase-2 was inhibited by the radiation. A CO2 incubator connected by optical fibers to a reflector telescope for collecting the solar light was built at Syowa station by the 49th Japanese Antarctica Research Expedition. The direction of the telescope was continuously controlled by a sun-tracker to follow the movement of the Sun automatically. The intensity of the collected light was monitored by a portable spectrophotometer housed inside. The human skin fibroblast cells were incubated in the CO2 chamber to investigate the effect of the solar radiation at Syowa station and were compared with those reference experiments at a laboratory in Japan. The results showed cell damage by strong UV radiation. The production of matrix metalloproteinase-2 was prompted by the moderate UV-B, but was inhibited by the strong UV-B radiation, as studied under laboratory conditions in Japan. The effect of strong solar radiation at Syowa station involving the radiation of UV-B region was estimated to be of the same extent of the radiation caused by an artificial UV-B light with the intensity more than 50 mJ/cm2.

  18. Prevention of experimental autoimmune encephalomyelitis in DA rats by grafting primary skin fibroblasts engineered to express transforming growth factor-beta1.

    Science.gov (United States)

    Zargarova, T; Kulakova, O; Prassolov, V; Zharmukhamedova, T; Tsyganova, V; Turobov, V; Ivanov, D; Parfenov, M; Sudomoina, M; Chernajovsky, Y; Favorova, O

    2004-08-01

    To determine whether primary fibroblasts producing latent transforming growth factor beta1 (TGF-beta1) are capable of down-regulating experimental autoimmune encephalomyelitis (EAE), a retroviral vector TGF-beta1-pBabe-neo (-5'UTR) was used for efficient gene transfer into primary skin fibroblasts of DA rats. After heat activation, conditioned medium from the transduced fibroblasts was found to inhibit significantly in vitro proliferation of lymphocytes from lymph nodes of DA rats with EAE. Intraperitoneal administration of TGF-beta1-transduced fibroblasts into DA rats during the priming phase of EAE resulted in a significant reduction in mortality and in the mean clinical and EAE scores versus the control immunized animals treated with non-transduced fibroblasts.

  19. Prevention of experimental autoimmune encephalomyelitis in DA rats by grafting primary skin fibroblasts engineered to express transforming growth factor-β1

    Science.gov (United States)

    Zargarova, T; Kulakova, O; Prassolov, V; Zharmukhamedova, T; Tsyganova, V; Turobov, V; Ivanov, D; Parfenov, M; Sudomoina, M; Chernajovsky, Y; Favorova, O

    2004-01-01

    To determine whether primary fibroblasts producing latent transforming growth factor β1 (TGF-β1) are capable of down-regulating experimental autoimmune encephalomyelitis (EAE), a retroviral vector TGF-β1-pBabe-neo (−5′UTR) was used for efficient gene transfer into primary skin fibroblasts of DA rats. After heat activation, conditioned medium from the transduced fibroblasts was found to inhibit significantly in vitro proliferation of lymphocytes from lymph nodes of DA rats with EAE. Intraperitoneal administration of TGF-β1-transduced fibroblasts into DA rats during the priming phase of EAE resulted in a significant reduction in mortality and in the mean clinical and EAE scores versus the control immunized animals treated with non-transduced fibroblasts. PMID:15270848

  20. One-stage, simultaneous skin grafting with artificial dermis and basic fibroblast growth factor successfully improves elasticity with maturation of scar formation.

    Science.gov (United States)

    Hamuy, Rodrigo; Kinoshita, Naoshi; Yoshimoto, Hiroshi; Hayashida, Kenji; Houbara, Seiji; Nakashima, Masahiro; Suzuki, Keiji; Mitsutake, Norisato; Mussazhanova, Zhanna; Kashiyama, Kazuya; Hirano, Akiyoshi; Akita, Sadanori

    2013-01-01

    The efficacy of one-stage artificial dermis and skin grafting was tested in a nude rat model. Reconstruction with artificial dermis is usually a two-stage procedure with 2- to 3-week intermission. If one-stage use of artificial dermis and split-thickness skin grafting are effective, the overall burden on patients and the medical cost will markedly decrease. The graft take rate, contraction rate, tissue elasticity, histology, morphometric analysis of the dermal thickness, fibroblast counting, immunohistochemistry of α-smooth muscle actin, matrix metalloproteinase-2, CD31, and F4/80, as well as gelatin zymography, real-time reverse transcriptase polymerase chain reaction for matrix metalloproteinase-2, and electron microscopy, were investigated from day 3 to 3 months postoperatively. The graft take rate was good overall in one-stage artificial dermis and skin grafting groups up to 3 weeks, and the contraction rate was greater in the two-staged artificial dermis and skin grafting group than in the skin grafting alone or one stage of artificial dermis and skin grafting groups. Split-thickness skin grafting with artificial dermis and basic fibroblast growth factor at a concentration of 1 μg/cm(2) showed significantly greater elasticity by Cutometer, and the dermal thickness was significantly thinner, fibroblast counting was significantly greater, and the α-smooth muscle actin expression level was more notable with a more mature blood supply in the dermis and more organized dermal fibrils by electron microscopy at 3 weeks. Thus, one-stage artificial dermis and split-thickness skin grafting with basic fibroblast growth factor show a high graft take rate and better tissue elasticity determined by Cutometer analysis, maturity of the dermis, and increased fibroblast number and blood supply compared to a standard two-stage reconstruction.

  1. Comparison of the effect of cortisol on aromatase activity and androgen metabolism in two human fibroblast cell lines derived from the same individual

    DEFF Research Database (Denmark)

    Svenstrup, B; Brünner, N; Dombernowsky, P

    1990-01-01

    The effect of preincubation with cortisol on estrogen and androgen metabolism was investigated in human fibroblast monolayers grown from biopsies of genital and non-genital skin of the same person. The activity in the cells of aromatase, 5 alpha-reductase, 17 beta-hydroxysteroid oxidoreductase.......5-1.0 x 10(-6) M in both cell lines. When preincubation with cortisol was omitted no estrogen synthesis was detected. The formation of androgen was not altered after preincubation with cortisol. Pronounced differences were found in estrogen and in androgen metabolism in the two cell lines suggesting...... a local regulation of the hormonal environment. The aromatase activity, which is low in many tissues could be stimulated by cortisol without altering the androgen metabolism was found to be a suitable system for investigations of the cellular interconversion of androgens and estrogens...

  2. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence

    OpenAIRE

    Yafan Yang; Shuangshuang Li

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect U...

  3. Generation of iPSC line iPSC-FH2.1 in hypoxic conditions from human foreskin fibroblasts

    Directory of Open Access Journals (Sweden)

    María Questa

    2016-03-01

    Full Text Available Human foreskin fibroblasts were used to generate the iPSC line iPSC-FH2.1 using the EF1a-hSTEMCCA-loxP vector expressing OCT4, SOX2, c-MYC and KLF4, in 5% O2 culture conditions. Stemness was confirmed, as was pluripotency both in vivo and in vitro, in normoxia and hypoxia. Human Embryonic Stem Cell (hESC line WA-09 and reprogrammed fibroblast primary culture HFF-FM were used as controls.

  4. Dermal fibroblast expression of stromal cell-derived factor-1 (SDF-1) promotes epidermal keratinocyte proliferation in normal and diseased skin.

    Science.gov (United States)

    Quan, Chunji; Cho, Moon Kyun; Shao, Yuan; Mianecki, Laurel E; Liao, Eric; Perry, Daniel; Quan, Taihao

    2015-12-01

    Stromal cells provide a crucial microenvironment for overlying epithelium. Here we investigated the expression and function of a stromal cell-specific protein, stromal cell-derived factor-1 (SDF-1), in normal human skin and in the tissues of diseased skin. Immunohistology and laser capture microdissection (LCM)-coupled quantitative real-time RT-PCR revealed that SDF-1 is constitutively and predominantly expressed in dermal stromal cells in normal human skin in vivo. To our surprise, an extremely high level of SDF-1 transcription was observed in the dermis of normal human skin in vivo, evidenced by much higher mRNA expression level than type I collagen, the most abundant and highly expressed protein in human skin. SDF-1 was also upregulated in the tissues of many human skin disorders including psoriasis, basal cell carcinoma (BCC), and squamous cell carcinoma (SCC). Double immunostaining for SDF-1 and HSP47 (heat shock protein 47), a marker of fibroblasts, revealed that fibroblasts were the major source of stroma-cell-derived SDF-1 in both normal and diseased skin. Functionally, SDF-1 activates the ERK (extracellular-signal-regulated kinases) pathway and functions as a mitogen to stimulate epidermal keratinocyte proliferation. Both overexpression of SDF-1 in dermal fibroblasts and treatment with rhSDF-1 to the skin equivalent cultures significantly increased the number of keratinocyte layers and epidermal thickness. Conversely, the stimulative function of SDF-1 on keratinocyte proliferation was nearly completely eliminated by interfering with CXCR4, a specific receptor of SDF-1, or by knock-down of SDF-1 in fibroblasts. Our data reveal that extremely high levels of SDF-1 provide a crucial microenvironment for epidermal keratinocyte proliferation in both physiologic and pathologic skin conditions.

  5. Enzyme-Treated Asparagus Extract Attenuates Hydrogen Peroxide-Induced Matrix Metalloproteinase-9 Expression in Murine Skin Fibroblast L929 Cells.

    Science.gov (United States)

    Shirato, Ken; Takanari, Jun; Ogasawara, Junetsu; Sakurai, Takuya; Imaizumi, Kazuhiko; Ohno, Hideki; Kizaki, Takako

    2016-05-01

    Enzyme-treated asparagus extract (ETAS) exerts a wide variety of beneficial biological actions including facilitating anti-cortisol stress and neurological anti-aging responses. However, the anti-skin aging effects of ETAS remain to be elucidated. Reactive oxygen species (ROS) play pivotal roles in skin aging. Increased ROS levels in fibroblasts in response to ultraviolet irradiation activate c-Jun N-terminal kinase (JNK) and its downstream transcription factor activator protein-1 (AP-1), and the resultant gene expression of matrix metalloproteinase (MMP) isoforms accelerates collagen breakdown in the dermis. Therefore, we explored whether ETAS has anti-skin aging effects by attenuating the oxidative stress responses in fibroblasts. Simultaneous treatment of murine skin L929 fibroblasts with hydrogen peroxide (H2O2) and either ETAS or dextrin showed that ETAS significantly suppressed H2O2-induced expression of MMP-9 mRNA as measured by real-time polymerase chain reaction. ETAS also clearly suppressed H2O2-stimulated phosphorylation of c-Jun (AP-1 subunit) and JNK as determined by Western blot. However, ETAS did not affect the increased amounts of carbonyl proteins in response to H2O2, also as determined by Western blotting. These results suggest that ETAS diminishes cellular responsiveness to ROS but does not scavenge ROS. Thus, ETAS has the potential to prevent skin aging through attenuating the oxidative stress responses in dermal fibroblasts.

  6. Non-saponifiable fraction of cocoa shell butter: effect on rat and human skin fibroblasts.

    Science.gov (United States)

    Warocquier-Clerout, R; Sigot, M; Ouraghi, M; Chaveron, H

    1992-02-01

    Synopsis Non-saponifiable lipid fraction (ICSB) extracted from cocoa shell butter was solubilized in dimethylformamide (DMF) and analysed for its biological activity on growth of rat and human fibroblasts. Non-saponifiables (10 mug ml(-1)) partially protected cells from toxicity of DMF (1%) and allowed the growth of fibroblasts cultivated in optimal conditions (10% fetal calf serum-FCS, 37 degrees C) or improved the survival of cells maintained in altered conditions (2.5% FCS, 35 degrees C). At higher concentration (ICSB 50 mug ml(-1), DMF 1%), the protective effect was suppressed. ICSB was fractionated by chromatography into four compounds: sterols, terpenic alcohols, tocopherols and hydrocarbons +/- carotenoids. We found that biological activity of ICSB was mostly due to the major fraction containing sterols.

  7. Interaction of cambial dermal cells (fibroblasts) and epidermis in morphofunctional area of mouse skin.

    Science.gov (United States)

    Yavisheva, T M; Shcherbakov, S D; Golubeva, I S; Sharafutdinov, G Z

    2007-11-01

    Epidermis and dermis form an integral morphofunctional area, where cambial cells proliferate and their first-division daughter cells differentiate. An important feature of this area is different rates of the development of daughter cells (fibroblasts) in the dermis and epidermis, which is greater in the epidermis. This asymmetry results in the prevalence of first epidermal daughter cells and, hence, their effect on cambial cells, and then of stromal daughter cells and their effects on cambial cells. The regulator factors of epidermal daughter cells promote unblocking of the major polarity axis of cambial (mother) cell, while stromal cells (fibroblasts) induce their polarization along the major axis and the onset of mitosis. In the dermis and epidermis, division of cambial cells is asymmetric; a prominent role in the formation of mother and daughter cells is given to the basal membrane as an elastic support. Mother and daughter cells form ring-like structures generating electric field that can promote differentiation of the daughter cells.

  8. Cytoprotective effects of mild plasma-activated medium against oxidative stress in human skin fibroblasts

    Science.gov (United States)

    Horiba, Minori; Kamiya, Tetsuro; Hara, Hirokazu; Adachi, Tetsuo

    2017-01-01

    Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues and has emerged as a novel technology for medical applications. NTAPP affects cells not only directly, but also indirectly with previously prepared plasma-activated medium (PAM). The objective of this study was to demonstrate the preconditioning effects of “mild PAM” which was prepared under relatively mild conditions, on fibroblasts against cellular injury generated by a high dose of hydrogen peroxide (H2O2). We observed the preconditioning effects of mild PAM containing approximately 50 μM H2O2. Hydrogen peroxide needs to be the main active species in mild PAM for it to exert preconditioning effects because the addition of catalase to mild PAM eliminated these effects. The nuclear translocation and recruitment of nuclear factor erythroid 2-related factor 2 (Nrf2) to antioxidant response elements (ARE) in heme oxygenase 1 (HO-1) promoters and the up-regulation of HO-1 were detected in fibroblasts treated with mild PAM. The addition of ZnPP, a HO-1-specific inhibitor, or the knockdown of Nrf2 completely abrogated the preconditioning effects. Our results demonstrate that mild PAM protects fibroblasts from oxidative stress by up-regulating HO-1, and the H2O2-induced activation of the Nrf2-ARE pathway needs to be involved in this reaction. PMID:28169359

  9. Cholesteatoma fibroblasts promote epithelial cell proliferation through overexpression of epiregulin.

    Directory of Open Access Journals (Sweden)

    Mamoru Yoshikawa

    Full Text Available To investigate whether keratinocytes proliferate in response to epiregulin produced by subepithelial fibroblasts derived from middle ear cholesteatoma. Tissue samples were obtained from patients undergoing tympanoplasty. The quantitative polymerase chain reaction and immunohistochemistry were performed to examine epiregulin expression and localization in cholesteatoma tissues and retroauricular skin tissues. Fibroblasts were cultured from cholesteatoma tissues and from normal retroauricular skin. These fibroblasts were used as feeder cells for culture with a human keratinocyte cell line (PHK16-0b. To investigate the role of epiregulin in colony formation by PHK16-0b cells, epiregulin mRNA expression was knocked down in fibroblasts by using short interfering RNA and epiregulin protein was blocked with a neutralizing antibody. Epiregulin mRNA expression was significantly elevated in cholesteatoma tissues compared with that in normal retroauricular skin. Staining for epiregulin was more intense in the epithelial cells and subepithelial fibroblasts of cholesteatoma tissues than in retroauricular skin. When PHK16-0b cells were cultured with cholesteatoma fibroblasts, their colony-forming efficiency was 50% higher than when these cells were cultured with normal skin fibroblasts. Also, knockdown of epiregulin mRNA in cholesteatoma fibroblasts led to greater suppression of colony formation than knockdown in skin fibroblasts. Furthermore, the colony-forming efficiency of PHK16-0b cells was significantly reduced after treatment with an epiregulin neutralizing antibody in co-culture with cholesteatoma fibroblasts, but not in co-culture with skin fibroblasts. These results suggest that keratinocyte hyperproliferation in cholesteatoma is promoted through overexpression of epiregulin by subepithelial fibroblasts via epithelial-mesenchymal interactions, which may play a crucial role in the pathogenesis of middle ear cholesteatoma.

  10. Toxicity evaluation of ZnO nanostructures on L929 fibroblast cell line using MTS assay

    Energy Technology Data Exchange (ETDEWEB)

    Bakhori, Siti Khadijah Mohd; Mahmud, Shahrom; Ann, Ling Chuo [Nano-optoelectronics Research and Technology Laboratory (NOR.), School of Physics, Universiti Sains Malaysia, 11800, USM, Pulau Pinang (Malaysia); Mohamed, Azman Seeni; Saifuddin, Siti Nazmin [Integrative Medicine Cluster, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Bandar Putra Bertam, 13200 Kepala Batas, Pulau Pinang (Malaysia); Masudi, Sam’an Malik; Mohamad, Dasmawati [Craniofacial Science Laboratory, School of Dentistry, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan (Malaysia)

    2015-04-24

    ZnO has wide applications in medical and dentistry apart from being used as optoelectronic devices such as solar cells, photodetectors, sensors and light emitting diodes (LEDs). Therefore, the toxicity evaluation is important to know the toxicity level on normal cell line. The toxicity of two grades ZnO nanostructures, ZnO-4 and ZnO-8 have been carried out using cytotoxicity test of MTS assay on L929 rat fibroblast cell line. Prior to that, ZnO-4 and ZnO-8 were characterized for its morphology, structure and optical properties using FESEM, X-ray diffraction, and Photoluminescence respectively. The two groups revealed difference in morphology and exhibit slightly shifted of near band edge emission of Photoluminescence other than having a similar calculated crystallite size of nanostructures. The viability of cells after 72h were obtained and the statistical significance value was calculated using SPSS v20. The p value is more than 0.05 between untreated and treated cell with ZnO. This insignificant value of p>0.05 can be summarized as a non-toxic level of ZnO-4 and ZnO-8 on the L929 cell line.

  11. Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

    Energy Technology Data Exchange (ETDEWEB)

    Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.; Miller, A.D.

    1987-02-01

    Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

  12. Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts.

    Science.gov (United States)

    Maeda-Sano, Katsura; Gotoh, Mari; Morohoshi, Toshiro; Someya, Takao; Murofushi, Hiromu; Murakami-Murofushi, Kimiko

    2014-09-01

    Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR1, Gi/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR1-Gi/o followed by the PI3K, ERK, and CREB signaling pathway.

  13. Effect of capping agents on the cytotoxicity of silver nanoparticles in human normal and cancer skin cell lines

    Science.gov (United States)

    Netchareonsirisuk, Ponsawan; Puthong, Songchan; Dubas, Stephan; Palaga, Tanapat; Komolpis, Kittinan

    2016-11-01

    Silver nanoparticles (AgNPs) are among the most widely used nanomaterials in medical and consumer products. However, safety in the uses of AgNPs is still controversial. The toxicity of AgNPs toward various cell types has been reported to depend on the surface properties of the nanoparticles. In this study, the effect of AgNPs with the average size of 5-15 nm on the viability of the CCD-986SK human normal skin fibroblast cell line and A375 human malignant melanoma cell line was evaluated. Comparative toxicity studies, based on MTT assay, were performed by using either sodium alginate or poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (PSSMA) as capping agent in the nanoparticle preparation. The cytotoxicity tests revealed that AgNO3 alone was highly toxic to both cell types while both alginate and PSSMA alone were not toxic. AgNPs capped with alginate were selectively toxic to the cancer cell line but not to the normal cell line while AgNPs capped with PSSMA were toxic to both cancer and normal cell lines. Judging from the 50 % inhibition concentration (IC50), it was found that the cancer cell line was more sensitive to AgNPs than the normal cell line. Study on the mode of cell death by annexin V and propidium iodide staining revealed that AgNPs induced more apoptotic cell death (84-90 %) than necrosis (8-12 %) in the skin cancer cell line. These results suggest that the toxicity of AgNPs depended on the type of capping agent and the type of cell line.

  14. Cysteine-rich protein 61 (CCN1) domain-specific stimulation of matrix metalloproteinase-1 expression through αVβ3 integrin in human skin fibroblasts.

    Science.gov (United States)

    Qin, Zhaoping; Fisher, Gary J; Quan, Taihao

    2013-04-26

    Human skin largely comprises collagenous extracellular matrix. The hallmark of skin aging is fragmentation of collagen fibrils. Matrix metalloproteinases (MMPs) are largely responsible for collagen degradation. MMP-1, principally derived from dermal fibroblasts, is the major protease capable of initiating degradation of native fibrillar collagens. Presently, we report that CCN1, a secreted and extracellular matrix-associated protein, is elevated in aged human skin dermal fibroblasts in vivo and stimulates MMP-1 expression through functional interaction with αVβ3 integrin in human dermal fibroblasts. CCN1 contains four conserved structural domains. Our results indicate that the three N-terminal domains (IGFBP, VWC, and TSP1), but not the C-terminal CT domain, are required for CCN1 to stimulate MMP-1 expression. This stimulation is dependent on interaction between the active structural domains and αVβ3 integrin. The interaction of VWC domain with integrin αVβ3 is necessary and requires functional cooperation with adjacent IGFBP and TSP1 domains to stimulate MMP-1 expression. Finally, induction of MMP-1 expression in dermal fibroblasts by CCN1 N-terminal domains resulted in fragmentation of type I collagen fibrils in a three-dimensional collagen lattice model. These data suggest that domain-specific interactions of CCN1 with αVβ3 integrin contribute to human skin aging by stimulating MMP-1-mediated collagen fibril fragmentation.

  15. Tamarind seed coat extract restores reactive oxygen species through attenuation of glutathione level and antioxidant enzyme expression in human skin fibroblasts in response to oxidative stress

    Directory of Open Access Journals (Sweden)

    Oranuch Nakchat

    2014-05-01

    Conclusions: TSCE exhibited antioxidant activities by scavenging ROS, attenuating GSH level that could protect human skin fibroblast cells from oxidative stress. Our results highlight the antioxidant mechanism of tamarind seed coat through an antioxidant enzyme system, the extract potentially benefits for health food and cosmeceutical application of tamarind seed coat.

  16. Recovery of fibroblast-like cells from refrigerated goat skin up to 41 d of animal death.

    Science.gov (United States)

    Okonkwo, Charles; Singh, Mahipal

    2015-05-01

    Successful cloning of animals using somatic cell nuclear transfer requires undamaged nuclear DNA from desired donor cell types. In vitro culture of cells is one way of ensuring nuclear integrity. The goal of this study was to evaluate the limits of postmortem cell survival/culture in refrigerated goat ear skin tissues which could be used for long-term storage and cloning of animals in future. To achieve this, 60 explants from 6 different goats were cultured after 0, 3, 6, 9, 13, 16, 20, 23, 27, 30, 33, 37, and 41 d postmortem and observed under inverted microscope for outgrowth of fibroblast-like cells, after 10-12 d of culture. Explants from all time points including 19% from 41-dpm tissues exhibited outgrowth. However, the percentage of outgrowth positive explants, as well as culture confluence, reduced with increasing postmortem time interval. Cell cultures established from primary outgrowth of 41-dpm tissues when compared for their growth profile with similarly obtained 0-dpm cultures revealed similar growth curve and cell morphology. Cytogenetic analysis of 41-dpm tissue-derived cell populations revealed a normal female karyotype with 60 XX homologous chromosomes indicating genetic stability of the cell population. In conclusion, these results show that refrigerated skin tissue remains alive for more than a month and that the cells derived from such tissues are normal and can be cryopreserved for long-term storage and future cloning of animals with desired genetics.

  17. The impact of nitrite and antioxidants on ultraviolet-A-induced cell death of human skin fibroblasts.

    Science.gov (United States)

    Opländer, Christian; Cortese, Miriam M; Korth, Hans-Gert; Kirsch, Michael; Mahotka, Csaba; Wetzel, Wiebke; Pallua, Norbert; Suschek, Christoph V

    2007-09-01

    Nitrite (NO(2)(-)) occurs ubiquitously in biological fluids such as blood and sweat. Ultraviolet A-induced nitric oxide formation via decomposition of cutaneous nitrite, accompanied by the production of reactive oxygen (ROS) or nitrogen species (RNS), represents an important source for NO in human skin physiology. Examining the impact of nitrite and the antioxidants glutathione (GSH), Trolox (TRL), and ascorbic acid (ASC) on UVA-induced toxicity of human skin fibroblasts (FB) we found that NO(2)(-) concentration-dependently enhances the susceptibility of FB to the toxic effects of UVA by a mechanism comprising enhanced induction of lipid peroxidation. While ASC completely protects FB cultures from UVA/NO(2)(-)-induced cell damage, GSH or TRL excessively enhances UVA/NO(2)(-)-induced cell death by a mechanism comprising nitrite concentration-dependent TRL radical formation or GSH-derived oxidative stress. Simultaneously, in the presence of GSH or TRL the mode of UVA/NO(2)(-)-induced cell death changes from apoptosis to necrosis. In summary, during photodecomposition of nitrite, ROS or RNS formation may act as strong toxic insults. Although inhibition of oxidative stress by NO and other antioxidants represents a successful strategy for protection from UVA/NO(2)(-)-induced injuries, GSH and TRL may nitrite-dependently aggravate the injurious impact by TRL or GSH radical formation, respectively.

  18. Antiprion activity of cholesterol esterification modulators: a comparative study using ex vivo sheep fibroblasts and lymphocytes and mouse neuroblastoma cell lines.

    Science.gov (United States)

    Pani, Alessandra; Norfo, Claudia; Abete, Claudia; Mulas, Claudia; Putzolu, Marirosa; Laconi, Sergio; Orrù, Christina Doriana; Cannas, M Dolores; Vascellari, Sarah; La Colla, Paolo; Dessì, Sandra

    2007-11-01

    Our studies on the role of cholesterol homeostasis in the pathogenesis of scrapie revealed abnormal accumulation of cholesterol esters in ex vivo peripheral blood mononuclear cells (PBMCs) and skin fibroblasts from healthy and scrapie-affected sheep carrying a scrapie-susceptible genotype compared to sheep with a resistant genotype. Similar alterations were observed in mouse neuroblastoma N2a cell lines persistently infected with mouse-adapted 22L and RML strains of scrapie that showed up to threefold-higher cholesterol ester levels than parental N2a cells. We now report that proteinase K-resistant prion protein (PrPres)-producing cell populations of subclones from scrapie-infected cell lines were characterized by higher cholesterol ester levels than clone populations not producing PrPres. Treatments with a number of drugs known to interfere with different steps of cholesterol metabolism strongly reduced the accumulation of cholesterol esters in ex vivo PBMCs and skin fibroblasts from scrapie-affected sheep but had significantly less or no effect in their respective scrapie-resistant or uninfected counterparts. In scrapie-infected N2a cells, inhibition of cholesterol esters was associated with selective antiprion activity. Effective antiprion concentrations of cholesterol modulators (50% effective concentration [EC(50)] range, 1.4 to 40 microM) were comparable to those of antiprion reference compounds (EC(50) range, 0.6 to 10 microM). These data confirm our hypothesis that abnormal accumulation of cholesterol esters may represent a biological marker of susceptibility to prion infection/replication and a novel molecular target of potential clinical importance.

  19. Protective effects of APP 17-mer peptide on cultured human skin fibroblasts after irradiation with ultraviolet light%APP17肽通过抑制细胞内ROS保护紫外线照射后人皮肤成纤维细胞

    Institute of Scientific and Technical Information of China (English)

    陈慧; 连石; 朱威

    2011-01-01

    Objective Ultraviolet light (UV) is known to cause photoaging of skin.UV irradiation can damage proliferation capacity and induce collagenase in fibroblasts in the dermis .Many researchers have explored the potential photo-protective agents;however,no ideal agent has been widely accepted .Amyloid precursor protein 17-mer peptide (APP17-mer peptide),an active peptide segment,has been reported to be responsible for the trophic effect in clonal CNS neuronal line ,fibroblast cell line and HaCat cells.The aim of this study was to explore the effects of APP17-mer peptide on cultured fibroblasts after ultraviolet irradiation .Methods Human skin fibroblasts were cultured in DMEM medium with or without APP 17-mer peptide (concentrations ranging from 20 μmol/L,40 μmol/L,to 80 μmol/L).The cultured fibroblasts were exposed to a single UV irradiation,and the proliferation activity of fibroblasts was detected by a MTT assay .The ex-pression of matrix metalloproteinase-1 (MMP-1) mRNA was analyzed quantitatively following real -time RT-PCR.The generation of intracellular reactive oxygen species (ROS) was measured with fluorescent quantita-tion method.Results A single exposure to UV irradiation depressed proliferation activity of fibroblasts com -pared with sham-irradiated control (P <0.05).40 μmol/L and 80 μmol/L APP17-mer peptide increased the cellular proliferation activity in UV irradiated and unirradiated fibroblasts (P <0.05),however,20 μmol/L did not show such protective effects (P >0.05).A single exposure of fibroblasts to UV irradiation resulted in 1.78 fold up-regulation of MMP-1 mRNA compared with unirradiated sample (P <0.05),and 40 μmol/L and 80 μmol/L APP17-mer peptide decreased the expression of MMP -1 mRNA (P <0.05 and P <0.01,re-spectively).UV irradiation increased generation of ROS in cultured fibroblasts (P <0.05).40 μmol/L APP17-mer peptide inhibited the generation of ROS in irradiated fibroblasts .Conclusions APP17-mer pep-tide can

  20. Cationic star-shaped polymer as an siRNA carrier for reducing MMP-9 expression in skin fibroblast cells and promoting wound healing in diabetic rats

    Directory of Open Access Journals (Sweden)

    Li N

    2014-07-01

    Full Text Available Na Li,1,* Heng-Cong Luo,1,* Chuan Yang,1 Jun-Jie Deng,2 Meng Ren,1 Xiao-Ying Xie,1 Diao-Zhu Lin,1 Li Yan,1 Li-Ming Zhang2 1Department of Endocrinology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, People’s Republic of China; 2DSAPM Lab and PCFM Lab, Institute of Polymer Science, Department of Polymer and Materials Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, People’s Republic of China *These authors contributed equally to this work Background: Excessive expression of matrix metalloproteinase-9 (MMP-9 is deleterious to the cutaneous wound-healing process in the context of diabetes. The aim of the present study was to explore whether a cationic star-shaped polymer consisting of ß-cyclodextrin (ß-CD core and poly(amidoamine dendron arms (ß-CD-[D3]7 could be used as the gene carrier of small interfering RNA (siRNA to reduce MMP-9 expression for enhanced diabetic wound healing. Methods: The cytotoxicity of ß-CD-(D37 was investigated by 3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay (MMT method in the rat CRL1213 skin fibroblast cell line. The transfection efficiency of ß-CD-(D37/MMP-9-small interfering RNA (siRNA complexes was determined by confocal microscopy and flow cytometry. Quantitative real time (RT polymerase chain reaction was performed to measure the gene expression of MMP-9 after the transfection by ß-CD-(D37/MMP-9-siRNA complexes. The ß-CD-(D37/MMP-9-siRNA complexes were injected on the wounds of streptozocin-induced diabetic rats. Wound closure was measured on days 4 and 7 post-wounding. Results: ß-CD-(D37 exhibited low cytotoxicity in fibroblast cells, and easily formed the complexes with MMP-9-siRNA. The ß-CD-(D37/MMP-9-siRNA complexes were readily taken up by fibroblast cells, resulting in the downregulation of MMP-9 gene expression (P<0.01. Animal experiments revealed that the treatment by ß-CD-(D37/MMP-9-siRNA complexes enhanced wound

  1. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence.

    Science.gov (United States)

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption.

  2. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence

    Directory of Open Access Journals (Sweden)

    Yafan Yang

    2015-01-01

    Full Text Available Ultraviolet (UV irradiation causes damage in skin by generating excessive reactive oxygen species (ROS and induction of matrix metalloproteinases (MMPs, leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption.

  3. Dandelion Extracts Protect Human Skin Fibroblasts from UVB Damage and Cellular Senescence

    Science.gov (United States)

    Yang, Yafan; Li, Shuangshuang

    2015-01-01

    Ultraviolet (UV) irradiation causes damage in skin by generating excessive reactive oxygen species (ROS) and induction of matrix metalloproteinases (MMPs), leading to skin photoageing. Dandelion extracts have long been used for traditional Chinese medicine and native American medicine to treat cancers, hepatitis, and digestive diseases; however, less is known on the effects of dandelion extracts in skin photoageing. Here we found that dandelion leaf and flower extracts significantly protect UVB irradiation-inhibited cell viability when added before UVB irradiation or promptly after irradiation. Dandelion leaf and flower extracts inhibited UVB irradiation-stimulated MMP activity and ROS generation. Dandelion root extracts showed less action on protecting HDFs from UVB irradiation-induced MMP activity, ROS generation, and cell death. Furthermore, dandelion leaf and flower but not root extracts stimulated glutathione generation and glutathione reductase mRNA expression in the presence or absence of UVB irradiation. We also found that dandelion leaf and flower extracts help absorb UVB irradiation. In addition, dandelion extracts significantly protected HDFs from H2O2-induced cellular senescence. In conclusion, dandelion extracts especially leaf and flower extracts are potent protective agents against UVB damage and H2O2-induced cellular senescence in HDFs by suppressing ROS generation and MMP activities and helping UVB absorption. PMID:26576225

  4. Phosphoproteomics Profiling of Human Skin Fibroblast Cells Reveals Pathways and Proteins Affected by Low Doses of Ionizing Radiation

    Science.gov (United States)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-01-01

    Background High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. Principal Findings We have identified 7117 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conclusions Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health. PMID:21152398

  5. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Miller, John H.; Gritsenko, Marina A.; Zhao, Rui; Du, Xiuxia; Livesay, Eric A.; Purvine, Samuel O.; Monroe, Matthew E.; Wang, Yingchun; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2010-11-30

    Background: High doses of ionizing radiation result in biological damage, however the precise relationships between long term health effects, including cancer, and low dose exposures remain poorly understood and are currently extrapolated using high dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose dependent responses to radiation. Principle Findings: We have identified 6845 unique phosphopeptides (2566 phosphoproteins) from control and irradiated (2 and 50 cGy) primary human skin fibroblasts one hour post-exposure. Dual statistical analyses based on spectral counts and peak intensities identified 287 phosphopeptides (from 231 proteins) and 244 phosphopeptides (from 182 proteins) that varied significantly following exposure to 2 and 50 cGy respectively. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatics analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role of MAP kinase and protein kinase A (PKA) signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. Conlcusions: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provides a basis for the systems level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at

  6. Phosphoproteomics profiling of human skin fibroblast cells reveals pathways and proteins affected by low doses of ionizing radiation.

    Directory of Open Access Journals (Sweden)

    Feng Yang

    Full Text Available BACKGROUND: High doses of ionizing radiation result in biological damage; however, the precise relationships between long-term health effects, including cancer, and low-dose exposures remain poorly understood and are currently extrapolated using high-dose exposure data. Identifying the signaling pathways and individual proteins affected at the post-translational level by radiation should shed valuable insight into the molecular mechanisms that regulate dose-dependent responses to radiation. PRINCIPAL FINDINGS: We have identified 7117 unique phosphopeptides (2566 phosphoproteins from control and irradiated (2 and 50 cGy primary human skin fibroblasts 1 h post-exposure. Semi-quantitative label-free analyses were performed to identify phosphopeptides that are apparently altered by radiation exposure. This screen identified phosphorylation sites on proteins with known roles in radiation responses including TP53BP1 as well as previously unidentified radiation-responsive proteins such as the candidate tumor suppressor SASH1. Bioinformatic analyses suggest that low and high doses of radiation affect both overlapping and unique biological processes and suggest a role for MAP kinase and protein kinase A (PKA signaling in the radiation response as well as differential regulation of p53 networks at low and high doses of radiation. CONCLUSIONS: Our results represent the most comprehensive analysis of the phosphoproteomes of human primary fibroblasts exposed to multiple doses of ionizing radiation published to date and provide a basis for the systems-level identification of biological processes, molecular pathways and individual proteins regulated in a dose dependent manner by ionizing radiation. Further study of these modified proteins and affected networks should help to define the molecular mechanisms that regulate biological responses to radiation at different radiation doses and elucidate the impact of low-dose radiation exposure on human health.

  7. The Apoptotic Effects of the P300 Activator on Breast Cancer and Lung Fibroblast Cell Lines

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Salahshoor

    2013-10-01

    Full Text Available Background: P300 is an enzyme that acetylates histones during stress. It alsoacetylates several non-histone proteins, including P53 which is the most important tumorsuppressor gene. P53 plays an important role in the apoptosis of tumor cells. Hereby,this study describes the potency of cholera toxin B subunit as a P300 activator to induceapoptosis in a breast cancer cell line (MCF-7 and a lung fibroblast cell line (MRC-5as a non-tumorigenic control sample. Methods: MCF-7 and MRC-5 were cultured in RPMI-1640 and treated with orwithout cholera toxin B subunit at the concentration of 85.43 μmol/L, based on the half-maximal inhibitory concentration index at different times (24, 48 and 72 h. Thepercentage of apoptotic cells was measured by flow cytometry. Real-time quantitativeRT-PCR was performed to estimate the mRNA expression of P300 in MCF-7 and MRC-5 with cholera toxin B subunit at different times. We used the ELISA and Bradford proteintechniques to detect levels of total and acetylated P53 protein generated in MCF-7 andMRC-5. Results: Our findings indicated that the cholera toxin B subunit effectively andsignificantly induced more apoptosis in MCF-7 compared to MRC-5. We showed thatexpression of P300 up-regulated by increasing the time of the cholera toxin B subunittreatment in MCF-7 but not in MRC-5. In addition, the acetylated and total P53protein levels increased more in MCF-7 cells than in MRC-5 cells.Conclusion: Cholera toxin B subunit induced significant cell death in MCF-7, butit could be well tolerated in MRC-5. Therefore, cholera toxin B subunit can besuggested as an anti-cancer agent.

  8. Transcriptome comparison of human neurons generated using induced pluripotent stem cells derived from dental pulp and skin fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jian Chen

    Full Text Available Induced pluripotent stem cell (iPSC technology is providing an opportunity to study neuropsychiatric disorders through the capacity to grow patient-specific neurons in vitro. Skin fibroblasts obtained by biopsy have been the most reliable source of cells for reprogramming. However, using other somatic cells obtained by less invasive means would be ideal, especially in children with autism spectrum disorders (ASD and other neurodevelopmental conditions. In addition to fibroblasts, iPSCs have been developed from cord blood, lymphocytes, hair keratinocytes, and dental pulp from deciduous teeth. Of these, dental pulp would be a good source for neurodevelopmental disorders in children because obtaining material is non-invasive. We investigated its suitability for disease modeling by carrying out gene expression profiling, using RNA-seq, on differentiated neurons derived from iPSCs made from dental pulp extracted from deciduous teeth (T-iPSCs and fibroblasts (F-iPSCs. This is the first RNA-seq analysis comparing gene expression profiles in neurons derived from iPSCs made from different somatic cells. For the most part, gene expression profiles were quite similar with only 329 genes showing differential expression at a nominally significant p-value (p<0.05, of which 63 remained significant after correcting for genome-wide analysis (FDR <0.05. The most striking difference was the lower level of expression detected for numerous members of the all four HOX gene families in neurons derived from T-iPSCs. In addition, an increased level of expression was seen for several transcription factors expressed in the developing forebrain (FOXP2, OTX1, and LHX2, for example. Overall, pathway analysis revealed that differentially expressed genes that showed higher levels of expression in neurons derived from T-iPSCs were enriched for genes implicated in schizophrenia (SZ. The findings suggest that neurons derived from T-iPSCs are suitable for disease

  9. The ontogeny of scarless healing II: EGF and PDGF-B gene expression in fetal rat skin and fibroblasts as a function of gestational age.

    Science.gov (United States)

    Peled, Z M; Rhee, S J; Hsu, M; Chang, J; Krummel, T M; Longaker, M T

    2001-10-01

    Twenty years ago, surgeons noted the ability of early-gestation fetal skin to heal in a scarless manner. Since that time, numerous investigators have attempted to elucidate the mechanisms behind this phenomenon. As a result of this effort, it is now well established that many animals undergo a transition late in development from scarless cutaneous healing to a scar-forming, adultlike phenotype. The authors have been interested in the role played by cytokines known to be involved in the adult wound-healing process and how they relate to scarless repair. They therefore asked the following question: Are genes for epidermal growth factor (EGF) and platelet-derived growth factor-B (PDGF-B) expressed differentially as a function of gestational age in fetal rat skin and dermal fibroblasts? To answer this question, skin from fetal Sprague-Dawley rats (N = 56) at time points that represented both the scarless and scar-forming periods of rat gestation was harvested. In addition, fibroblasts derived from fetal rat skin were cultured in vitro at similar times. These cells were expanded in culture and, when confluent, total ribonucleic acid from both fibroblasts and whole skin was extracted and subjected to Northern blot analysis with probes for EGF and PDGF-B. Results demonstrated that neither EGF nor PDGF-B gene expression changed markedly as a function of gestational age in fetal fibroblasts alone. In whole skin, however, both EGF and PDGF-B demonstrated a marked decrease in gene expression with increasing gestational age. Furthermore, the most striking decrease in gene expression for both cytokines came between 16 and 18 days of gestation-the transition point between scarless and scar-forming repair in the fetal rat. These data suggest that EGF and PDGF may play a role in the mechanism of scarless cutaneous repair. Moreover, it appears that fetal fibroblasts are not the cell type responsible for this differential gene expression. These results raise questions about the

  10. A New Skin Tensiometer Device: Computational Analyses To Understand Biodynamic Excisional Skin Tension Lines

    OpenAIRE

    Paul, Sharad P.; Justin Matulich; Nick Charlton

    2016-01-01

    One of the problems in planning cutaneous surgery is that human skin is anisotropic, or directionally dependent. Indeed, skin tension varies between individuals and at different body sites. Many a surgeon has tried to design different devices to measure skin tension to help plan excisional surgery, or to understand wound healing. However, many of the devices have been beset with problems due to many confounding variables - differences in technical ability, material (sutures) used and variabil...

  11. Alpha-1,3-galactosyltransferase-deficient miniature pigs produced by serial cloning using neonatal skin fibroblasts with loss of heterozygosity

    Science.gov (United States)

    Kim, Young June; Ahn, Kwang Sung; Kim, Minjeong; Kim, Min Ju; Ahn, Jin Seop; Ryu, Junghyun; Heo, Soon Young; Park, Sang-Min; Kang, Jee Hyun; Choi, You Jung; Shim, Hosup

    2017-01-01

    Objective Production of alpha-1,3-galactosyltransferase (αGT)-deficient pigs is essential to overcome xenograft rejection in pig-to-human xenotransplantation. However, the production of such pigs requires a great deal of cost, time, and labor. Heterozygous αGT knockout pigs should be bred at least for two generations to ultimately obtain homozygote progenies. The present study was conducted to produce αGT-deficient miniature pigs in much reduced time using mitotic recombination in neonatal ear skin fibroblasts. Methods Miniature pig fibroblasts were transfected with αGT gene-targeting vector. Resulting gene-targeted fibroblasts were used for nuclear transfer (NT) to produce heterozygous αGT gene-targeted piglets. Fibroblasts isolated from ear skin biopsies of these piglets were cultured for 6 to 8 passages to induce loss of heterozygosity (LOH) and treated with biotin-conjugated IB4 that binds to galactose-α-1,3-galactose, an epitope produced by αGT. Using magnetic activated cell sorting, cells with monoallelic disruption of αGT were removed. Remaining cells with LOH carrying biallelic disruption of αGT were used for the second round NT to produce homozygous αGT gene-targeted piglets. Results Monoallelic mutation of αGT gene was confirmed by polymerase chain reaction in fibroblasts. Using these cells as nuclear donors, three heterozygous αGT gene-targeted piglets were produced by NT. Fibroblasts were collected from ear skin biopsies of these piglets, and homozygosity was induced by LOH. The second round NT using these fibroblasts resulted in production of three homozygous αGT knockout piglets. Conclusion The present study demonstrates that the time required for the production of αGT-deficient miniature pigs could be reduced significantly by postnatal skin biopsies and subsequent selection of mitotic recombinants. Such procedure may be beneficial for the production of homozygote knockout animals, especially in species, such as pigs, that require a

  12. Photobiomodulation of distinct lineages of human dermal fibroblasts: a rational approach towards the selection of effective light parameters for skin rejuvenation and wound healing

    Science.gov (United States)

    Mignon, Charles; Uzunbajakava, Natallia E.; Raafs, Bianca; Moolenaar, Mitchel; Botchkareva, Natalia V.; Tobin, Desmond J.

    2016-03-01

    Distinct lineages of human dermal fibroblasts play complementary roles in skin rejuvenation and wound healing, which makes them a target of phototherapy. However, knowledge about differential responses of specific cell lineages to different light parameters and moreover the actual molecular targets remain to be unravelled. The goal of this study was to investigate the impact of a range of parameters of light on the metabolic activity, collagen production, and cell migration of distinct lineages of human dermal fibroblasts. A rational approach was used to identify parameters with high therapeutic potential. Fibroblasts exhibited both inhibitory and cytotoxic change when exposed to a high dose of blue and cyan light in tissue culture medium containing photo-reactive species, but were stimulated by high dose red and near infrared light. Cytotoxic effects were eliminated by refreshing the medium after light exposure by removing potential ROS formed by extracellular photo-reactive species. Importantly, distinct lineages of fibroblasts demonstrated opposite responses to low dose blue light treatment when refreshing the medium after exposure. Low dose blue light treatment also significantly increased collagen production by papillary fibroblasts; high dose significantly retarded closure of the scratch wound without signs of cytotoxicity, and this is likely to have involved effects on both cell migration and proliferation. We recommend careful selection of fibroblast subpopulations and their culture conditions, a systematic approach in choosing and translating treatment parameters, and pursuit of fundamental research on identification of photoreceptors and triggered molecular pathways, while seeking effective parameters to address different stages of skin rejuvenation and wound healing.

  13. Multiangle light scattering flow photometry of cultured human fibroblasts: comparison of normal cells with a mutant line containing cytoplasmic inclusions.

    Science.gov (United States)

    Schafer, I A; Jamieson, A M; Petrelli, M; Price, B J; Salzman, G C

    1979-01-01

    Multi-angle light scattering flow photometry was used to study the light scattering properties of normal cultured fibroblasts and a mutant fibroblast line containing cytoplasmic lysosomal inclusions. The effect of glutaraldehyde fixation on the light scattering properties of the cells was also examined and correlated with their ultrastructure. Normal fibroblasts showed uniform organelle distribution with few vacuoles or dense bodies in the cytoplasm while the mutant line showed abnormal cytoplasmic inclusions of varying morphology, density and lucency. As predicted by light scattering theory, the mutant cells containing the cytoplasmic inclusions scattered more light at large angles (greater than theta = 1.85 degrees) than did the normal cells. Glutaraldehyde fixation decreased light scattering at small angles (less than theta = 1.85 degrees), increased light scattering at larger angles (greater than theta = 1.85 degrees) in both normal and mutant cells and enhanced resolution of the light scattering signatures. The mutant line scattered 2-3 times more light at a wide angle (greater than theta = 12.74 degrees) than did the normal cells. These data suggest that abnormal lysosomal storage inclusion bodies in the cytoplasm of the cells can be detected by differential light scattering methods.

  14. The effect of Centella asiatica, vitamins, glycolic acid and their mixtures preparations in stimulating collagen and fibronectin synthesis in cultured human skin fibroblast.

    Science.gov (United States)

    Hashim, Puziah

    2014-03-01

    Centella asiatica (Linn.) Urban is well known in promoting wound healing and provides significant benefits in skin care and therapeutic products formulation. Glycolic acid and vitamins also play a role in the enhancement of collagen and fibronectin synthesis. Here, we evaluate the specific effect of Centella asiatica (CA), vitamins, glycolic acid and their mixture preparations to stimulate collagen and fibronectin synthesis in cultured human fibroblast cells. The fibroblast cells are incubated with CA, glycolic acid, vitamins and their mixture preparations for 48 h. The cell lysates were analyzed for protein content and collagen synthesis by direct binding enzyme immunoassay. The fibronectin of the cultured supernatant was measured by sandwich enzyme immunoassay. The results showed that CA, glycolic acid, vitamins A, E and C significantly stimulate collagen and fibronectin synthesis in the fibroblast. Addition of glycolic acid and vitamins to CA further increased the levels of collagen and fibronectin synthesis to 8.55 and 23.75 μg/100 μg, respectively. CA, glycolic acid, vitamins A, E, and C, and their mixtures demonstrated stimulatory effect on both extra-cellular matrix synthesis of collagen and fibronectin in in vitro studies on human foreskin fibroblasts, which is beneficial to skin care and therapeutic products formulation.

  15. Autophagy in human skin fibroblasts: Comparison between young and aged cells and evaluation of its cellular rhythm and response to Ultraviolet A radiation.

    Science.gov (United States)

    Pernodet, Nadine; Dong, Kelly; Pelle, Edward

    2016-01-01

    Autophagic mechanisms play critical roles in cell maintenance. Damaged organelles that are not removed by autophagosomes, which act by engulfing and degrading these cellular components, have been linked to various pathologies. Recently, the progression of aging has also been correlated to a compromised autophagic response. Here, we report for the first time a significant reduction in autophagic levels in synchronized aged normal human skin fibroblasts as compared to young fibroblasts. We measured a 77.9% reduction in autophagy as determined by reverse transcription-polymerase chain reaction for LC3B expression, a microtubule-associated protein correlated to late stage autophagosome formation. In addition, we visualized these same changes by immunocytofluorescence with antibodies directed against LC3B. By harvesting synchronized, as well as unsynchronized cells over time, we were also able to measure for the first time a nighttime peak in autophagy that was present in young but absent in aged fibroblasts. Finally, since human skin is constantly subjected to environmentally induced oxidative stress from sunlight, we exposed fibroblasts to 10 J/cm2 ultraviolet A and found, in good agreement with current literature, not only that irradiation could partially reactivate autophagy in the aged cells, but also that this increase was phase shifted earlier from its endogenous temporal pattern because of its loss of synchronization with circadian rhythm.

  16. Redox-dependent induction of antioxidant defenses by phenolic diterpenes confers stress tolerance in normal human skin fibroblasts: Insights on replicative senescence.

    Science.gov (United States)

    Carvalho, Ana C; Gomes, Andreia C; Pereira-Wilson, Cristina; Lima, Cristovao F

    2015-06-01

    Mild stress-induced hormesis represents a promising strategy for targeting the age-related accumulation of molecular damage and, therefore, for preventing diseases and achieving healthy aging. Fruits, vegetables, and spices contain a wide variety of hormetic phytochemicals, which may explain the beneficial health effects associated with the consumption of these dietary components. In the present study, the induction of cellular antioxidant defenses by the phenolic diterpenes carnosic acid (CA) and carnosol (CS) were studied in normal human skin fibroblasts, and insights into the aging process at the cellular level investigated. We observed that CA and CS induced several cytoprotective enzymes and antioxidant defenses in human fibroblasts, whose induction was dependent on the cellular redox state for CS and associated with Nrf2 signaling for both compounds. The stress response elicited by preincubation with CS conferred a cytoprotective action against a following oxidant challenge with tert-butyl hydroperoxide, confirming its hormetic effect. Preincubation of normal fibroblasts with CS also protected against hydrogen peroxide-induced premature senescence. Furthermore, cultivation of middle passage normal human skin fibroblasts in the presence of CS ameliorated the physiological state of cells during replicative senescence. Our results support the view that mild stress-induced antioxidant defenses by CS can confer stress tolerance in normal cells and may have important implications in the promotion of healthy aging.

  17. Curcumin induces stress response and hormetically modulates wound healing ability of human skin fibroblasts undergoing ageing in vitro.

    Science.gov (United States)

    Demirovic, Dino; Rattan, Suresh I S

    2011-10-01

    Wound healing becomes impaired in several diseases and during ageing. A commonly used model for the study of wound healing is a scratched monolayer of cells in vitro, which is convenient for the analysis of the cellular and molecular changes occurring during the two phases of wound healing, namely cell migration and cell proliferation. Cell migration, which is the primary event to occur during initial wound healing, is inversely dependent on the number of focal adhesions (FA) that attach cells to the extracellular matrix. Here we report that the number of FA, measured by determining the levels of FA-proteins paxillin and talin, increase with increasing population doubling level of the serially passaged normal adult skin fibroblasts, and that this increase may account for the age-related slowing down of wound healing in vitro. We also report that curcumin, a component of the widely used spice turmeric, modulates wound healing in vitro in a biphasic dose response manner, being stimulatory at low doses (between 1 and 5 μM), and inhibitory at higher doses. Furthermore, our results show that the hormetic effects of low levels of curcumin are achieved by virtue of it being a hormetin in terms of the induction of stress response pathways, including Nrf2 and HO-1 in human cells.

  18. Low doses of nanodiamonds and silica nanoparticles have beneficial hormetic effects in normal human skin fibroblasts in culture.

    Science.gov (United States)

    Mytych, Jennifer; Wnuk, Maciej; Rattan, Suresh I S

    2016-04-01

    Nanodiamonds (ND) and silica nanoparticles (SiO2-NP) have been much investigated for their toxicity at high doses, little is known about their biological activity at low concentrations. Here we report the biphasic dose response of ND and SiO2-NP in modulating normal human facial skin fibroblasts (FSF1) in culture. ND and SiO2-NP at low concentration (up to 0.5 μg/ml) had beneficial effects on FSF1 in terms of increasing their proliferation and metabolic activity. Exposure of FSF1 cells to low levels of NP enhanced their wound healing ability in vitro and slowed down aging during serial passaging as measured by maintenance of youthful morphology, reduction in the rate of loss of telomeres, and the over all proliferative characteristics. Furthermore, NP treatment induced the activation of Nrf2- and FOXO3A-mediated cellular stress responses, including an increased expression of heme oxygenease (HO-1), sirtuin (SIRT1), and DNA methyltransferase II (DNMT2). These results imply that ND and SiO2-NP at low doses are potential hormetins, which exert mild stress-induced beneficial hormetic effects through improved survival, longevity, maintenance, repair and function of human cells.

  19. Tamarind seed coat extract restores reactive oxygen species through attenuation of glutathione level and antioxidant enzyme expression in human skin fibroblasts in response to oxidative stress

    OpenAIRE

    Oranuch Nakchat; Nonthaneth Nalinratana; Duangdeun Meksuriyen; Sunanta Pongsamart

    2014-01-01

    Objective: To investigate the role and mechanism of tamarind seed coat extract (TSCE) on normal human skin fibroblast CCD-1064Sk cells under normal and oxidative stress conditions induced by hydrogen peroxide (H2O2). Methods: Tamarind seed coats were extracted with boiling water and then partitioned with ethyl acetate before the cell analysis. Effect of TSCE on intracellular reactive oxygen species (ROS), glutathione (GSH) level, antioxidant enzymes such as superoxide dismutase (SOD), glut...

  20. Parapoxvirus orf virus infection induces an increase in interleukin-8, tumour necrosis factor-α, and decorin in goat skin fibroblast cells

    Directory of Open Access Journals (Sweden)

    Wang Lingling

    2016-09-01

    Full Text Available Introduction: Orf virus (ORFV is a prototype Parapoxvirus species in the Poxviridae family that causes serious zoonotic infectious disease. Goat skin fibroblast (GSF cells are the major host targets of ORFV. Interleukin 8 (IL-8 and tumour necrosis factor (TNF-α are known to play a vital role in immune response during viral infections. However, the manner of variation over time of their level of expression in GSF cells remains unclear.

  1. Establishment of a pig fibroblast-derived cell line for locus-directed transgene expression in cell cultures and blastocysts

    DEFF Research Database (Denmark)

    Jakobsen, Jannik E; Li, Juan; Moldt, Brian;

    2011-01-01

    We report the establishment of a spontaneously immortalized pig cell line designated Pig Flip-in Visualize (PFV) for locus-directed transgene expression in pig cells and blastocysts. The PFV cell line was isolated from pig ear fibroblasts transfected with a Sleeping Beauty DNA transposon......-based docking vector harbouring a selection gene, an eGFP reporter gene, and an Flp recombinase site for locus-directed gene insertion. PFV cells have insertion of a single docking vector with stable eGFP expression and generated phenotypic normal blastocysts with transgene expression after somatic cell nuclear...

  2. Effects of plant sterols derived from Aloe vera gel on human dermal fibroblasts in vitro and on skin condition in Japanese women

    Directory of Open Access Journals (Sweden)

    Tanaka M

    2015-02-01

    Full Text Available Miyuki Tanaka,1 Eriko Misawa,1 Koji Yamauchi,1 Fumiaki Abe,1 Chiaki Ishizaki2 1Functional Food Research Department, Food Science and Technology Institute, Morinaga Milk Industry Co, Ltd, Zama, Kanagawa, 2Ebisu Skin Research Center, Inforward, Inc., Tokyo, Japan Background: Aloe is known for its topical use for treating wounds and burns. Many previous studies reported the healing effects of Aloe vera. However, there are few clinical studies on the effect of orally administered A. vera gel on the skin. Aloe sterols are a type of plant sterols that have the capability to regulate the metabolism of glucose and lipids. In a recent study, we confirmed that ingested Aloe sterols reached the peripheral tissues through the bloodstream. However, their influence on dermal fibroblasts has not been investigated. Methods: First, we investigated the capability of Aloe sterols (cycloartenol and lophenol to stimulate human dermal fibroblasts in vitro. Then, we investigated the effect of intake of Aloe vera gel powder (AVGP containing 40 µg Aloe sterols on the skin conditions in Japanese women with dry skin in a randomized, double-blind, placebo-controlled trial. Results: After cocultivation with Aloe sterols, the production of collagen and hyaluronic acid increased by approximately two-fold and 1.5-fold, and gene expression levels of these enzymes responsible for their synthesis were also observed in human dermal fibroblasts. An increase in arm skin hydration was observed at 8 weeks in the AVGP group, whereas a slight decrease in arm skin hydration was noted in the placebo group. However, there was no statistical difference between AVGP and placebo groups in skin moisture. In subgroup analysis, the change in the mean wrinkle depth was significantly lower in the AVGP group than in the control group. In addition, percent body fat after 8 weeks was significantly lower in the AVGP group. No AVGP intake-dependent harmful phenomenon was observed during the intake

  3. Differential translocation of heat shock factor-1 after mild and severe stress to human skin fibroblasts undergoing aging in vitro.

    Science.gov (United States)

    Demirovic, Dino; de Toda, Irene Martinez; Nizard, Carine; Rattan, Suresh I S

    2014-12-01

    Repeated exposure to mild heat shock (HS) has been shown to induce a wide range of health promoting hormetic effects in various biological systems, including human cells undergoing aging in vitro. In order to understand how cells distinguish between mild and severe stress, we have investigated the extent of early and immediate HS response by analyzing the nuclear translocation of the transcription factor heat shock factor-1 (HSF1), in serially passaged normal adult human facial skin fibroblasts exposed to mild (41 °C) or severe (43 °C) HS. Cells respond differently when exposed to mild and severe HS at different passage levels in terms of the extent of HSF1 translocation. In early passage young cells there was a 5-fold difference between mild and severe HS in the extent of HSF1 translocation. However, in near senescent late passage cells, the difference between mild and severe stress in terms of the extent of HSF1 translocation was reduced to less than 2-fold. One of the reasons for this age-related attenuation of heat shock response is due to the fact there was a higher basal level of HSF1 in the nuclei of late passage cells, which is indicative of increased intrinsic stress during cellular aging. These observations are consistent with previously reported data that whereas repeated mild stress given at younger ages can slow down aging and increase the lifespan, the same level of stress given at older ages may not provide the same benefits. Therefore, elucidating the early and immediate steps in the induction of stress response can be useful in deciding whether a particular level of stress is potentially hormetically beneficial or not.

  4. CopA3 Peptide Prevents Ultraviolet-Induced Inhibition of Type-I Procollagen and Induction of Matrix Metalloproteinase-1 in Human Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Dong-Hee Kim

    2014-05-01

    Full Text Available Ultraviolet (UV exposure is well-known to induce premature aging, which is mediated by matrix metalloproteinase-1 (MMP-1 activity. A 9-mer peptide, CopA3 (CopA3 was synthesized from a natural peptide, coprisin, which is isolated from the dung beetle Copris tripartitus. As part of our continuing search for novel bioactive natural products, CopA3 was investigated for its in vitro anti-skin photoaging activity. UV-induced inhibition of type-I procollagen and induction of MMP-1 were partially prevented in human skin fibroblasts by CopA3 peptide in a dose-dependent manner. At a concentration of 25 μM, CopA3 nearly completely inhibited MMP-1 expression. These results suggest that CopA3, an insect peptide, is a potential candidate for the prevention and treatment of skin aging.

  5. Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration.

    Science.gov (United States)

    Ouwehand, Krista; Spiekstra, Sander W; Waaijman, Taco; Scheper, Rik J; de Gruijl, Tanja D; Gibbs, Susan

    2011-11-01

    In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1β transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

  6. Impaired colony-forming ability following. gamma. irradiation of skin fibroblasts from tuberous scierosis patients. [/sup 60/Co

    Energy Technology Data Exchange (ETDEWEB)

    Paterson, M.C.; Sell, B.M.; Smith, B.P.; Bech-Hansen, N.T.

    1982-05-01

    The radiosensitivity of cultured dermal fibroblasts from human subjects afflicted with tuberous sclerosis (TS), a hereditary neurocutaneous syndrome, was assessed by assaying loss of colony-forming ability in response to acute ..gamma..-ray exposure. Related to control strains from clinically normal donors, three cell lines (GM1635, GM1643, GM2333) from two affected patients displayed enhanced sensitivity to inactivation by /sup 60/Co ..gamma..-ray treatment, whether administered oxically (air-saturated) or hypoxically (N/sub 2/-gassed); a fourth strain (GM1644) from a third patient exhibited normal radiosensitivity under both treatment conditions. The post-..gamma..-irradiaton colony-forming ability of the three hypersensitive TS strains was intermediate between that of normal controls and that of strains from patients inheriting the radiotherapy-sensitive neurovascular disorder ataxia telangiectasia. The variability in the radioresponse of the TS stains (three sensitive and one normal) is not surprising, considering the widely recognized clinical heterogeneity in the disease. Our findings, aside from providing a laboratory marker for early (possible presymptomatic) detection of persons at high risk for TS, may lead to a better understanding of the origin and progressive development of this multifaceted syndrome.

  7. PMN Leukocytes and Fibroblasts Numbers on Wound Burn Healing on the Skin of White Rat after Administration of Ambonese Plantain Banana

    Directory of Open Access Journals (Sweden)

    Juniarti

    2012-04-01

    Full Text Available A study of ambonese plantain banana (Musa paradisiaca var sapientum Lamb treatment in burn wound healing on the skin of white rats (Rattus novergicus has been conducted. The wound healing of burn injuries was evaluated by counting the number of PMN leukocytes and fibroblasts at the 7th, 14th, and 21st days following the treatment. The study showed that the decrease in number of PMN leukocytes of subjects treated with ambonese plantain banana was relatively more significant compared to both negative and positive control (Bioplacenton ®. In contrast, an increasing number of fibroblasts was significantly demonstrated at the 14th and 21st days after treatment. In conclusion, ambonese plantain banana treatment in burn injuries will provide better results compared to both positive and negative controls.

  8. Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF

    Directory of Open Access Journals (Sweden)

    Coppock Donald L

    2008-12-01

    Full Text Available Abstract Background Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1 To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF on these cells. 2 To describe a methodology to create fibroblast cell lines from patients with chronic wounds. Methods Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. Results Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA Cell Repository. Conclusion We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

  9. Alternative splicing of fibroblast growth factor receptor 3 produces a secreted isoform that inhibits fibroblast growth factor-induced proliferation and is repressed in urothelial carcinoma cell lines.

    Science.gov (United States)

    Tomlinson, Darren C; L'Hôte, Corine G; Kennedy, Wendy; Pitt, Eva; Knowles, Margaret A

    2005-11-15

    Fibroblast growth factor receptors (FGFRs) are a family of receptor tyrosine kinases that play key roles in proliferation, differentiation, and tumorigenesis. FGFR3 was identified as the major family member expressed in both normal human urothelium and cultured normal human urothelial (NHU) cells and was expressed as the IIIb isoform. We also identified a splice variant, FGFR3 Delta8-10, lacking exons encoding the COOH-terminal half of immunoglobulin-like domain III and the transmembrane domain. Previous reports have assumed that this is a cancer-specific splice variant. We showed that FGFR3 Delta8-10 is a normal transcript in NHU cells and is translated, N-glycosylated, and secreted. Primary urothelium expressed high levels of FGFR3 transcripts. In culture, levels were reduced in actively proliferating cells but increased at confluence and as cells approached senescence. Cells overexpressing FGFR3 IIIb showed FGF1-induced proliferation, which was inhibited by the addition of FGFR3 Delta8-10. In bladder tumor cell lines derived from aggressive carcinomas, there were significant alterations in the relative expression of isoforms including an overall decrease in the proportion of FGFR3 Delta8-10 and predominant expression of FGFR3 IIIc in some cases. In summary, alternative splicing of FGFR3 IIIb in NHU cells represents a normal mechanism to generate a transcript that regulates proliferation and in bladder cancer, the ratio of FGFR3 isoforms is significantly altered.

  10. Establishment and characterization of fetal fibroblast cell lines for generating human lysozyme transgenic goats by somatic cell nuclear transfer.

    Science.gov (United States)

    Liu, Jun; Luo, Yan; Zheng, Liming; Liu, Qingqing; Yang, Zhongcai; Wang, Yongsheng; Su, Jianmin; Quan, Fusheng; Zhang, Yong

    2013-10-01

    This study was performed to qualify goat fetal fibroblast (GFF) cell lines for genetic modification and somatic cell nuclear transfer (SCNT) to produce human lysozyme (hLYZ) transgenic goats. Nine GFF cell lines were established from different fetuses, and the proliferative lifespan and chromosomal stability were analyzed. The results suggested that cell lines with a longer lifespan had stable chromosomes compared with those of cells lines with a shorter lifespan. According to the proliferative lifespan, we divided GFF cell lines into two groups: cell lines with a long lifespan (GFF1/2/7/8/9; group L) and cell lines with a short lifespan (GFF3/4/5/6; group S). Next, a hLYZ expression vector was introduced into these cell lines by electroporation. The efficiencies of colony formation, expansion in culture, and the quality of transgenic clonal cell lines were significant higher in group L than those in group S. The mean fusion rate and blastocyst rate in group L were higher than those in group S (80.3 ± 1.7 vs. 65.1 ± 4.2 % and 19.5 ± 0.6 vs. 15.1 ± 1.1 %, respectively, P embryos into the oviducts of recipient goats, three live kids were born. PCR and Southern blot analyses confirmed integration of the transgene in cloned goats. In conclusion, the lifespan of GFF cell lines has a major effect on the efficiency to produce transgenic cloned goats. Therefore, the proliferative lifespan of primary cells may be used as a criterion to characterize the quality of cell lines for genetic modification and SCNT.

  11. Ultraviolet irradiation induces CYR61/CCN1, a mediator of collagen homeostasis, through activation of transcription factor AP-1 in human skin fibroblasts.

    Science.gov (United States)

    Quan, Taihao; Qin, Zhaoping; Xu, Yiru; He, Tianyuan; Kang, Sewon; Voorhees, John J; Fisher, Gary J

    2010-06-01

    UV irradiation from the sun elevates the production of collagen-degrading matrix metalloproteinases (MMPs) and reduces the production of new collagen. This imbalance of collagen homeostasis impairs the structure and function of the dermal collagenous extracellular matrix (ECM), thereby promoting premature skin aging (photoaging). We report here that aberrant dermal collagen homeostasis in UV-irradiated human skin is mediated in part by a CCN-family member, cysteine-rich protein-61 (CYR61/CCN1). CYR61 is significantly elevated in acutely UV-irradiated human skin in vivo, and UV-irradiated human skin fibroblasts. Knockdown of CYR61 significantly attenuates UV irradiation-induced inhibition of type-I procollagen and upregulation of MMP-1. Determination of CYR61 mRNA and protein indicates that the primary mechanism of CYR61 induction by UV irradiation is transcriptional. Analysis of CYR61 proximal promoter showed that a sequence conforming to the consensus binding site for transcription factor activator protein-1 (AP-1) is required for promoter activity. UV irradiation increased the binding of AP-1-family members c-Jun and c-Fos to this AP-1 site. Furthermore, functional blockade of c-Jun or knockdown of c-Jun significantly reduced the UV irradiation-induced activation of CYR61 promoter and CYR61 gene expression. These data show that CYR61 is transcriptionally regulated by UV irradiation through transcription factor AP-1, and mediates altered collagen homeostasis that occurs in response to UV irradiation in human skin fibroblasts.

  12. Protection against TGF-β1-induced fibrosis effects of IL-10 on dermal fibroblasts and its potential therapeutics for the reduction of skin scarring.

    Science.gov (United States)

    Shi, Ji-Hong; Guan, Hao; Shi, Shan; Cai, Wei-Xia; Bai, Xiao-Zhi; Hu, Xiao-Long; Fang, Xiao-Bin; Liu, Jia-Qi; Tao, Ke; Zhu, Xiong-Xiang; Tang, Chao-Wu; Hu, Da-Hai

    2013-05-01

    Scarring, tightly associated with fibrosis, is a significant symptomatic clinical problem. Interleukin 10 (IL-10) has been identified as a candidate scar-improving therapy based on preclinical studies. However, the molecular mechanism of IL-10 in scar improvement is still uncertain. In this study, human dermal fibroblasts stimulated with TGF-β1 were treated with IL-10 to analyze the mRNA and some of proteins' expression levels of type I collagen (Col1), type III collagen (Col3), alpha-smooth muscle actin (α-SMA), matrix metalloproteinase-1 (MMP1), MMP2, MMP8 and tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2 by real-time PCR and Western blot, to observe α-SMA-positive fibroblasts by immunocytochemistry. The contracture and improvement of fibroblast-populated collagen lattice (FPCL) and a murine model of wound healing were used to evaluate the scar-improving effects by histological staining. The results showed that IL-10 can significantly down-regulate the mRNA and protein expression levels of Col1, Col3, α-SMA, and up-regulate the mRNA expression levels of MMP1 and MMP8, and decrease α-SMA-positive fibroblasts. FPCL analysis showed that the IL-10 (20 ng/ml) can significantly inhibit the contracture, improve the architecture of FPCL. Wounds injected with IL-10 demonstrated that the appearance of scar was improved, the wound margin of scarring was narrow, and the deposition of collagens (Col1 and Col3) in regenerated tissue was relieved. These results provide direct evidences that IL-10 has the inhibitory effects on the excessive deposition of extracellular matrix components and fibroblast-to-myofibroblast transition, and show that IL-10 has the potential therapy in prevention and reduction of skin scarring.

  13. Induction of cAMP-dependent protein kinase A activity in human skin fibroblasts and rat osteoblasts by extremely low-frequency electromagnetic fields

    Energy Technology Data Exchange (ETDEWEB)

    Thumm, S.; Glock, S.; Haemmerle, H. [Natural and Medical Sciences Institute Reutlingen, University of Tuebingen (NMI), Markwiesenstrasse 55, D-72770 Reutlingen (Germany); Loeschinger, M.; Rodemann, H.P. [Section of Radiobiology and Molecular Environmental Research, University of Tuebingen, Roentgenweg 11, D-72076 Tuebingen (Germany)

    1999-09-01

    Sinusoidal extremely low-frequency electromagnetic fields (ELF-EMF; 7-8 mT, 20 Hz) have already been shown to inhibit proliferation and to accelerate terminal differentiation of human skin fibroblasts in vitro. In order to elucidate the underlying processes of signal transduction, we analysed the activity of cAMP-dependent protein kinase (PKA). EMF exposure for 60 min resulted in an increased PKA activity in human skin fibroblasts (2-fold) and rat embryonic osteoblasts (1.7-fold). Long-term exposure for up to 7 days with a constant 1 h-on/1 h-off EMF exposure rhythm indicated a transient stimulation of PKA activity during the first two exposure rhythms followed by a decrease to the baseline levels of sham-exposed controls. Based on these results, we postulate that a modulation of proliferation and differentiation processes in cells of mesenchymal origin is triggered by an immediate and transient EMF-induced increase in PKA activity. (orig.)

  14. Nrf2 and NF-κB Signaling Pathways Contribute to Porphyra-334-Mediated Inhibition of UVA-Induced Inflammation in Skin Fibroblasts.

    Science.gov (United States)

    Ryu, Jina; Kwon, Mi-Jin; Nam, Taek-Jeong

    2015-07-31

    In this study, we examined the protective effects of porphyra-334 against UVA-irradiated cellular damage and elucidated the underlying mechanisms. Porphyra-334 prevented UVA-induced cell death and exhibited scavenging activities against intracellular oxidative stress induced by UVA irradiation in skin fibroblasts. We found that porphyra-334 significantly reduced the secretion and expression of IL-6 and TNF-α, reduced nuclear expression of Nuclear factor-κB (NF-κB), and sustained NF-E2-related factor 2 (Nrf2) activation. Further mechanism research revealed that porphyra-334 promoted the Nrf2 signaling pathway in UVA-irradiated skin fibroblasts. Our results show that the antioxidant effect of porphyra-334 is due to the direct scavenging of oxidative stress and its inhibitory effects on NF-κB-dependent inflammatory genes, such as IL-6 and TNF-κ. Therefore, we hypothesize that boosting the Nrf2- NF-κB-dependent response to counteract environmental stress is a promising strategy for the prevention of UVA-related damage.

  15. Tamarind seed coat extract restores reactive oxygen species through attenuation of glutathione level and antioxidant enzyme expression in human skin fibroblasts in response to oxidative stress

    Institute of Scientific and Technical Information of China (English)

    Oranuch Nakchat; Nonthaneth Nalinratana; Duangdeun Meksuriyen; Sunanta Pongsamart

    2014-01-01

    Objective:To investigate the role and mechanism of tamarind seed coat extract (TSCE) on normal human skin fibroblast CCD-1064Sk cells under normal and oxidative stress conditions induced by hydrogen peroxide (H2O2). Methods:Tamarind seed coats were extracted with boiling water and then partitioned with ethyl acetate before the cell analysis. Effect of TSCE on intracellular reactive oxygen species (ROS), glutathione (GSH) level, antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase activity including antioxidant protein expression was investigated. Results: TSCE significantly attenuated intracellular ROS in the absence and presence of H2O2 by increasing GSH level. In the absence of H2O2, TSCE significantly enhanced SOD and catalase activity but did not affected on GPx. Meanwhile, TSCE significantly increased the protein expression of SOD and GPx in H2O2-treated cells. Conclusions: TSCE exhibited antioxidant activities by scavenging ROS, attenuating GSH level that could protect human skin fibroblast cells from oxidative stress. Our results highlight the antioxidant mechanism of tamarind seed coat through an antioxidant enzyme system, the extract potentially benefits for health food and cosmeceutical application of tamarind seed coat.

  16. Mécano-Stimulation™ of the skin improves sagging score and induces beneficial functional modification of the fibroblasts: clinical, biological, and histological evaluations

    Directory of Open Access Journals (Sweden)

    Humbert P

    2015-02-01

    Full Text Available Philippe Humbert,1,2 Ferial Fanian,1,2 Thomas Lihoreau,1,2 Adeline Jeudy,1,2 Ahmed Elkhyat,1,2 Sophie Robin,3 Carol Courderot-Masuyer,3 Hélène Tauzin,3 Christine Lafforgue,1,2,4 Marek Haftek5 1Research and Studies Center on the Integument (CERT, Department of Dermatology, Clinical Investigation Center (CIC 1431, Besançon University Hospital; 2INSERM UMR1098, FED4234 IBCT, University of Franche-Comté, Besançon, France; 3SARL BIOEXIGENCE, Besançon, France; 4Dermopharmacology and Cosmetology Unit, University of Paris Sud, France; 5University of Lyon 1, EA4169, Experimental, clinical and therapeutic aspects of the skin barrier function, INSERM US7 – CNRS UMS3453, Lyon, France Background: Loss of mechanical tension appears to be the major factor underlying decreased collagen synthesis in aged skin. Numerous in vitro studies have shown the impact of mechanical forces on fibroblasts through mechanotransduction, which consists of the conversion of mechanical signals to biochemical responses. Such responses are characterized by the modulation of gene expression coding not only for extracellular matrix components (collagens, elastin, etc. but also for degradation enzymes (matrix metalloproteinases [MMPs] and their inhibitors (tissue inhibitors of metalloproteinases [TIMPs]. A new device providing a mechanical stimulation of the cutaneous and subcutaneous tissue has been used in a simple, blinded, controlled, and randomized study. Materials and methods: Thirty subjects (aged between 35 years and 50 years, with clinical signs of skin sagging, were randomly assigned to have a treatment on hemiface. After a total of 24 sessions with Mécano-Stimulation™, biopsies were performed on the treated side and control area for in vitro analysis (dosage of hyaluronic acid, elastin, type I collagen, MMP9; equivalent dermis retraction; GlaSbox®; n=10 and electron microscopy (n=10. Furthermore, before and after the treatment, clinical evaluations and self

  17. Cytotoxicity of Silver Nanoparticles in Human Embryonic Stem Cell-Derived Fibroblasts and an L-929 Cell Line

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2012-01-01

    Full Text Available Consensus about the toxicity of silver nanoparticles (Ag-NPs has not been reached, even though extensive attention has been paid to this issue. This confusion may be due to physicochemical factors of Ag-NPs and the cell model used for biological safety evaluation. In the present study, human embryonic stem cell-derived fibroblasts (EBFs, which have been considered a closer representative of the in vivo response, were used as a novel cell model to assess the cytotoxicity of Ag-NPs (~20 nm and ~100 nm in comparison with L-929 fibroblast cell line. Cell proliferation, cell cycle, apoptosis, p53 expression, and cellular uptake were examined. Results showed that Ag-NPs presented higher cytotoxicity to EBF than to L-929. EBF demonstrated a stronger capacity to ingest Ag-NPs, a higher G2/M arrest, and more upgraduated p53 expression after exposed to Ag-NPs for 48 h when compared with L-929. It could be concluded that EBF exhibited a more sensitive response to Ag-NPs compared with L-929 cells, indicating that EBF may be a valid candidate for cytotoxicity screening assays of nanoparticles.

  18. Establishing Primary Adult Fibroblast Cultures From Rodents

    Science.gov (United States)

    Seluanov, Andrei; Vaidya, Amita; Gorbunova, Vera

    2010-01-01

    The importance of using primary cells, rather than cancer cell lines, for biological studies is becoming widely recognized. Primary cells are preferred in studies of cell cycle control, apoptosis, and DNA repair, as cancer cells carry mutations in genes involved in these processes. Primary cells cannot be cultured indefinitely due to the onset of replicative senescence or aneuploidization. Hence, new cultures need to be established regularly. The procedure for isolation of rodent embryonic fibroblasts is well established, but isolating adult fibroblast cultures often presents a challenge. Adult rodent fibroblasts isolated from mouse models of human disease may be a preferred control when comparing them to fibroblasts from human patients. Furthermore, adult fibroblasts are the only available material when working with wild rodents where pregnant females cannot be easily obtained. Here we provide a protocol for isolation and culture of adult fibroblasts from rodent skin and lungs. We used this procedure successfully to isolate fibroblasts from over twenty rodent species from laboratory mice and rats to wild rodents such as beaver, porcupine, and squirrel. PMID:20972406

  19. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols.

    Science.gov (United States)

    Mei, Xin; Wu, Yuan-Yuan; Mao, Xiao; Tu, You-Ying

    2011-01-01

    Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene.

  20. Establishment of a Transgenic Zebrafish Line for Superficial Skin Ablation and Functional Validation of Apoptosis Modulators In Vivo

    OpenAIRE

    2011-01-01

    BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a pla...

  1. Expression of nerve growth factor p75 receptor and sortilin in the skin fibroblasts and scar fibroblasts%神经生长因子p75受体与sortilin在皮肤及瘢痕成纤维细胞中的表达**★

    Institute of Scientific and Technical Information of China (English)

    冯璋; 张芮; 冯永强; 周一冲; 王一兵

    2013-01-01

    healing, but there is less research for the low-affinity nerve growth factor receptor p75 and sortilin in fibroblasts, and no reports on whether there are differences in expression of p75 and sortilin in the scar fibroblasts and normal skin fibroblasts. OBJECTIVE: To study the expression of low-affility nerve growth factor receptor p75 and sortilin in the normal human skin fibroblasts and the human keloid fibroblasts. METHODS: The keloid fibroblasts and normal hunman skin fibroblasts were cultured in vitro, and the immortalized epithelial cells HaCaT were used as the positive control. The real-time PCR was used to detect the mRNA expression of the p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts, and western blot and immunocytochemical staining were used to detect the protein expression of p75 and sortilin. RESULTS AND CONCLUSION: The real-time PCR and western blot results showed that in the protein and mRNA levels, p75 and sortilin showed positive expression in the keloid fibroblasts and normal human skin fibroblasts, and there was no significant difference in the expression of p75 between keloid fibroblasts and normal human skin fibroblasts, and the expressions of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were significantly lower than those in HaCaT. There was no significant difference of p75 expression between keloid fibroblasts and normal human skin fibroblasts, and the expression of sortilin in the keloid fibroblasts was significantly lower than that in the normal human skin fibroblasts (P < 0.05). Immunocytochemical staining result showed that the expression of p75 and sortilin in the keloid fibroblasts and normal human skin fibroblasts were distributed in the membrane and cytoplasm. Precursor nerve growth factor combined with high-affinity p75 receptor could promote the apoptosis of the cells with the help of sortilin, and the expression of sortilin in the keloid fibroblasts was significantly lower than

  2. Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

    Science.gov (United States)

    Zay, Agnes; Choy, Francis Y M; Patrick, Chelsea; Sinclair, Graham

    2011-06-01

    The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to

  3. Comparison of gene expression of metallothioneins, ubiquitin and p53 in fibroblasts from lung and skin of rats of different age

    Directory of Open Access Journals (Sweden)

    Y. G. Kot

    2015-09-01

    Full Text Available We studied gene expression of five metallothioneins (MT 1-5, ubiquitin and protein p53 and their products in fibroblasts culture of the skin and lungs of white rats of different ages (2 weeks, 1, 3, and 24 months and determined its (metallothionein 1-5 types, ubiquitin, p53 product quantity. All these proteins are protective ones, but perform their functions by using different mechanisms. Metallothionein bind, transport and excrete ions of bivalent metals, ubiquitin controls the cleavage of the defective and short-lived proteins in the proteasome, protein p53 controls apoptosis, thus ensuring the genome stability. The similarity of age dynamics of gene expression of ubiquitin and MT of cells of both sources has been shown – maximum at 3 months. Expression of p53 gene has a difference: both in the skin and lungs expression increases up to 24 months. Product quantity of p53 has a minimum in the skin at 3 months and remains constant; in the lungs, this value has a maximum at 1 month.

  4. Antiageing Mechanisms of a Standardized Supercritical CO2 Preparation of Black Jack (Bidens pilosa L. in Human Fibroblasts and Skin Fragments

    Directory of Open Access Journals (Sweden)

    Gustavo Dieamant

    2015-01-01

    Full Text Available The use of topical retinoids to treat skin disorders and ageing can induce local reactions, while oral retinoids are potent teratogens and produce several unwanted effects. This way, efforts to explore complementary care resources should be supported. Based on this, we evaluate the antiageing effects of a supercritical CO2 extract from Bidens pilosa L. (BPE-CO2A containing a standardized multicomponent mixture of phytol, linolenic, palmitic, linoleic, and oleic acids. BPE-CO2A was assessed for its effects on human dermal fibroblasts (TGF-β1 and FGF levels using ELISA; collagen, elastin, and glycosaminoglycan by colorimetric assays, and mRNA expression of RXR, RAR, and EGFr by qRT-PCR and human skin fragments (RAR, RXR, collagen, elastin, and glycosaminoglycan by immunohistochemical analysis. Levels of extracellular matrix elements, TGF-β1 and FGF, and EGFr gene expression were significantly increased by BPE-CO2A. The modulation of RXR and RAR was positively demonstrated after the treatment with BPE-CO2A or phytol, a component of BPE-CO2A. The effects produced by BPE-CO2A were similar to or better than those produced by retinol and retinoic acid. The ability to stimulate extracellular matrix elements, increase growth factors, and modulate retinoid and rexinoid receptors provides a basis for the development of preparation containing BPE-CO2A as an antiageing/skin-repair agent.

  5. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase+catalase and vitamins E and C.

    Science.gov (United States)

    Anane, R; Creppy, E E

    2001-09-01

    Lipid peroxidation is one of the main manifestations of oxidative damage and has been found to play an important role in the toxicity and carcinogenicity of many xenobiotics. In the present study, we investigated the possible induction of lipid peroxidation by aluminium in human foreskin fibroblast cultures by assaying the malondialdehyde (MDA) produced inside the cells. The MDA-thiobarbituric acid (TBA) adduct was assayed by HPLC using fluorometric quantification after extraction in n-butanol. Lactate dehydrogenase (LDH) release was used as a marker of aluminium toxicity. MDA production was significantly increased after 24 h incubation with aluminium and paralleled LDH release. Superoxide dismutase (SOD)+catalase and vitamins C and E added in the culture medium as oxygen radical and free radical scavengers were efficient in preventing MDA production by aluminium, indicating that oxidative processes are one of the main pathways whereby this metal induces cytotoxicity. The latter is also largely prevented, thus confirming the link between oxidative stress induced by aluminium and its cytotoxicity in human skin fibroblasts.

  6. Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami (Cypriniformes:Cyprinidae)%Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami (Cypriniformes: Cyprinidae)

    Institute of Scientific and Technical Information of China (English)

    Xiaoai WANG; Junxing YANG; Xiaoyong CHEN; Xiaofu PAN

    2012-01-01

    Though Yunnan province contains some 562 known species of fish,no cell lines from any of these have been made available to date.To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami,a fish endemic to Fuxian Lake,Yunnan,China,we established and characterized the major features of a continuous cell line (AGF Ⅱ) from the caudal fin tissue of A.grahami.This AGF Ⅱ cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year.The cell line was maintained in DMEM/F12 supplemented with 10% FBS,with a cellular doubling time of 51.1 h.We continued with more experiments to optimize the culture and storage conditions,and found a variety of interesting results: cells could grow at temperature between 24 ℃ and 28 ℃,with the optimal temperature of 28 ℃.Likewise,the growth rate ofA.grahami fin cells increased when the FBS proportion increased from 5% to 20%,with the optimal growth at the concentrations of 20% FBS; cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15; DMSO is a better cryoprotectant than Glycerol,EG and MeOH for AGFⅡ cells with optimal concentration of 5% DMSO.Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52,with a modal peak at 48 chromosomes,accounting for 39.8% of all cells.Using the same primer pairs specific to mtDNA,the AGF Ⅱ cell sequences obtained by PCR were identical to those from muscle tissues ofA.grahami.Both chromosome analysis and PCR amplification confirmed the AGF Ⅱ cells were from A.grahami,also indicating that that current long-term artificial propagation ofA.grahami has been successful.Finally,we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid,bright fluorescent signals were observed,suggesting that this cell line may be suitable for use in transfection and future gene expression studies.

  7. Morphological features of iPS cells generated from Fabry disease skin fibroblasts using Sendai virus vector (SeVdp).

    Science.gov (United States)

    Kawagoe, Shiho; Higuchi, Takashi; Otaka, Manami; Shimada, Yohta; Kobayashi, Hiroshi; Ida, Hiroyuki; Ohashi, Toya; Okano, Hirotaka J; Nakanishi, Mahito; Eto, Yoshikatsu

    2013-08-01

    We generated iPS cells from human dermal fibroblasts (HDFs) of Fabry disease using a Sendai virus (SeVdp) vector; this method has been established by Nakanishi et al. for pathogenic evaluation. We received SeVdp vector from Nakanishi and loaded it simultaneously with four reprogramming factors (Klf4, Oct4, Sox2, and c-Myc) to HDFs of Fabry disease; subsequently, we observed the presence of human iPS-like cells. The Sendai virus nucleocapsid protein was not detected in the fibroblasts by RT-PCR analysis. Additionally, we confirmed an undifferentiated state, alkaline phosphatase staining, and the presence of SSEA-4, TRA-1-60, and TRA-1-81. Moreover, ultrastructural features of these iPS cells included massive membranous cytoplasmic bodies typical of HDFs of Fabry disease. Thus, we successfully generated human iPS cells from HDFs of Fabry disease that retained the genetic conditions of Fabry disease; also, these abnormal iPS cells could not be easily differentiated into mature cell types such as neuronal cells, cardiomyocytes, etc. because of a massive accumulation of membranous cytoplasmic bodies in lysosomes, possibly the persistent damages of intracellular architecture.

  8. 脂多糖对正常人皮肤成纤维细胞胶原合成的影响%Influence of lipopolysaccharide on the collagen synthesis of normal human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    纪少军; 万立; 李凤玉; 贾国洪; 闫永宏; 石军

    2012-01-01

    Objective: To investigate the influence of lipopolysaccharide ( LPS ) on the collagen synthesis of normal human skin fibroblasts and its relation with wound healing. Methods: Normal skin fibroblasts were exposed to LPS for a certain period of time. Then the collagen synthesis of fibroblasts in culture medium was measured with the method of pepsin digestion after incorporation of H-proline into stable, single-layered, confluent fibroblasts. Results: Collagen synthesis of normal skin fibroblasts increased when challenged with LPS at the concentration of 0.005 μg·ml‐1 , and the increase showed a concentration dependent manner. However, when the concentration of LPS was 0. 5 μg·ml‐1 ,the influence of LPS on the collagen synthesis of normal skin fibroblasts began to decrease; when the concentration of LPS reached 1.0 μg·ml‐1 , collagen synthesis of fibroblasts were inhibited. Conclusion: LPS could promote collagen synthesis of normal human skin fibroblasts within a certain extent, suggesting that LPS might play an important role in wound healing.%目的:研究脂多糖(lipopolysaccharide,LPS)对正常人皮肤成纤维细胞胶原合成的影响,以阐明LPS在皮肤创伤愈合中的可能作用.方法:体外培养正常人皮肤成纤维细胞,采用3H-脯氨酸掺入法观察不同浓度(0、0.005、0.01、0.05、0.1、0.5和1.0 μg·ml-1)的LPS对成纤维细胞胶原合成的影响.结果:LPS刺激浓度在0.005 μg·ml-1时,皮肤成纤维细胞胶原蛋白合成增加;随着刺激浓度增加,刺激作用增强;但当LPS浓度为0.5 μg·ml-1时,促胶原合成作用开始降低;当浓度达到1.0 μg·ml-1时则呈现抑制效应.结论:在一定浓度范围内LPS促进成纤维细胞胶原合成,表明LPS在皮肤创伤愈合中可能具有重要作用.

  9. Antagonism of phenanthrene cytotoxicity for human embryo lung fibroblast cell line HFL-I by green tea polyphenols

    Energy Technology Data Exchange (ETDEWEB)

    Mei Xin [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China); Key Laboratory of Horticultural Plant Growth Development and Biotechnology of Ministry of Agriculture, Zhejiang University, Hangzhou 310029 (China); Wu Yuanyuan; Mao Xiao [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China); Tu Youying, E-mail: youytu@zju.edu.c [Department of Tea Science, Zhejiang University, Hangzhou 310029 (China)

    2011-01-15

    Polycyclic aromatic hydrocarbons (PAHs) have been detected in some commercial teas around the world and pose a threat to tea consumers. However, green tea polyphenols (GTP) possess remarkable antioxidant and anticancer effects. In this study, the potential of GTP to block the toxicity of the model PAH phenanthrene was examined in human embryo lung fibroblast cell line HFL-I. Both GTP and phenanthrene treatment individually caused dose-dependent inhibition of cell growth. A full factorial design experiment demonstrated that the interaction of phenanthrene and GTP significantly reduced growth inhibition. Using the median effect method showed that phenanthrene and GTP were antagonistic when the inhibitory levels were less than about 50%. Apoptosis and cell cycle detection suggested that only phenanthrene affected cell cycle significantly and caused cell death; GTP lowered the mortality of HFL-I cells exposed to phenanthrene; However, GTP did not affect modulation of the cell cycle by phenanthrene. - Green tea polyphenols antagonised cytotoxicity of a low-ring PAH phenanthrene.

  10. Non-invasive evaluation of skin tension lines with elastic waves

    CERN Document Server

    Deroy, Claire; Alinden, Aidan Mc; Annaidh, Aisling Ni

    2016-01-01

    Background: Since their discovery by Karl Langer in the 19th Century, Skin Tension Lines (STLs) have been used by surgeons to decide the location and orientation of an incision. Although these lines are patient-specific, most surgeons rely on generic maps to determine their orientation. Beyond the imprecise pinch test, there remains no accepted method for determining STLs in vivo. Methods: (i) The speed of an elastic motion travelling radially on the skin of canine cadavers was measured with a commercial device called the Reviscometer. (ii) Similar to the original experiments conducted by Karl Langer, circular excisions were made on the skin and the geometric changes to the resulting wounds and excised samples were used to determine the orientation of STLs. Results: A marked anisotropy in the speed of the elastic wave travelling radially was observed. The orientation of the fastest wave was found to correlate with the orientation of the elongated wound (P<0.001, R^2 = 74%). Similarly, the orientation of fa...

  11. Trypanosoma acomys (Wenyon, 1909): continuous culturing with a mouse fibroblast cell-line (A9).

    Science.gov (United States)

    Abdallah, M A; Abdel-Hafez, S K; al-Yaman, F M

    1990-01-01

    The continuous culturing of Trypanosoma acomys in the presence of a murine areolar-adipose cell line (A9) was possible for the 1st time. The trypanosomes were cultured at 37 degrees C with A9 in DMEM supplemented with 20% heat inactivated fetal bovine serum, using an initial inoculum from primary cultures of lung or blood clots from infected spiny mice. The cultures were maintained for 115 days and underwent 15 passages before termination and cryopreservation. Using this culture system T. acomys subcultures were initiated from 3 different initial inocula (3 x 10(4), 1.5 x 10(5) and 7.4 x 10(5) parasites/ml) and growth curves revealed that the lowest inoculum gave the best growth pattern. This inoculum yielded a population doubling time of less than 12 h for 4 days, a high peak density of 7 x 10(6) parasites/ml and the most gradual decline compared to the other 2 inocula. Rosetting epimastigotes and nests of amastigotes were observed in close association with the feeder layer cells. Epimastigotes were the most predominant form in culture supernatants but other morphological forms observed included trypomastigotes and sphaeromastigotes.

  12. Anti-Aging Effects of the Hanwoo Leg Bone, Foot and Tail Infusions (HLI, HFI and HTI) on Skin Fibroblast

    OpenAIRE

    Seol, Ja young; Yoon, Ji Young; Jeong, Hee Sun; Joo, Nami; Choi, Soon Young

    2016-01-01

    Many researchers revealed that collagen contribute to maintaining the skin’s elasticity and inhibit wrinkling of skin. Korean native cattle (Hanwoo) bone (leg bone, foot and tail) infusion contains the various inorganic materials, collagen and chondroitin sulfate. All of this, a large quantity of collagen is included in Hanwoo infusion. Therefore, this study emphasized on the effects of collagen in the Hanwoo bone infusion. For the first time, Hanwoo bone infusions were directly added to the ...

  13. Exploring the Anticancer Activity of Grape Seed Extract on Skin Cancer Cell Lines A431

    Directory of Open Access Journals (Sweden)

    V. Mohansrinivasan

    2015-08-01

    Full Text Available In this study, grape seeds were extracted using ethyl acetate and petroleum ether by solvent-solvent extraction method. The phytochemical tests were performed to identify different phytochemical compounds present in the grape seed extract (GSE. Antibacterial activity of the GSE was determined using agar diffusion method against Gram- positive and Gram-negative bacteria. Gas chromatography-mass spectrometry (GC-MS and Fourier transform infrared spectroscopy (FTIR analysis was done to identify the presence of bioactive compounds and their functional groups. The GC-MS results revealed a total of four compounds, known to have potent activity against cancer cells, viz, squalene, the most potent compound found in ethyl acetate extract and diethyl phthalate, ethyl-9- cis -11- trans octadecadienoate and (R-(--14,-methyl-8-Hexadecyn-1-ol in petroleum ether extract. Cytotoxic activity of the GSE was observed against skin cancer cell lines A4321 using 3-(4, 5-dimethylthiazol-2-yl-2-5-diphenyl tetrazolium bromide MTT assay. The IC50 value of the GSE against A431 skin cancer cell line was 480 µg/mL. This is first such report against A4321 cell lines. The study gives the overall perception about importance of GSE in medicine and nutraceuticals purposes.

  14. Use of Amniotic Microparticles Coated With Fibroblasts Overexpressing SDF-1α to Create an Environment Conducive to Neovascularization for Repair of Full-Thickness Skin Defects.

    Science.gov (United States)

    Zhang, Yun-qing; Ji, Shi-zhao; Fang, He; Zheng, Yong-jun; Luo, Peng-fei; Wu, Hai-bin; Wu, Min-juan; Wang, Zhi-hong; Xiao, Shi-chu; Xia, Zhao-fan

    2016-01-01

    As angiogenesis and vasculogenesis involve the complex network structures of various types of cells, extracellular matrix components, and cytokines, it is still difficult to exactly mimic the microenvironment of vascularization in vivo. In our study, we constructed a complex containing highly proliferative fibroblasts that can secrete extracellular matrix components and growth factors to chemotaxize endothelial progenitor cells (EPCs) in an attempt to create an ideal microenvironment for quick vascularization. Amniotic membrane microparticles (mAM) rich in type IV collagen (COL IV) and laminin (LN) were prepared, and human dermal fibroblasts (HDF) were infected with lentivirus (LV) of overexpression of SDF-1α to construct SDF-1α(ov)HDF. Using the rotary cell culture system (RCCS), mAM was loaded with HDF or SDF-1α(ov)HDF to construct HDF-mAM and SDF-1α(ov)HDF-mAM complexes. The complexes were able to secrete various types of active peptides (IL-6, IL-8, TGF-β, and bFGF) during in vitro culture. In addition, SDF-1α(ov)HDF-mAM complex highly expressed SDF-1α. Transwell assay showed SDF-1α(ov)HDF-mAM complex had an apparent chemotactic effect on EPCs. Transplantation of complexes onto full-thickness skin defects of C57BL mice further demonstrated that SDF-1α expression and the number of peripheral EPCs at days 3, 5, and 7 in the SDF-1α(ov)HDF-mAM group were significantly higher than that in other groups (p SDF-1α(ov)HDF-mAM group was significantly higher than that in HDF-mAM group (p SDF-1α could chemotaxize EPCs to reach local wounds, thus further accelerating angiogenesis in the transplant site. The technique described may prove to be a new model for accelerating vascularization of tissue and organ transplants and chronic ischemic wounds.

  15. Cytotoxicity evaluation of a new fast set highly viscous conventional glass ionomer cement with L929 fibroblast cell line

    Directory of Open Access Journals (Sweden)

    Hany Mohamed Aly Ahmed

    2011-01-01

    Conclusions : This new fast set highly viscous conventional GIC showed low cytotoxicity to mouse fibroblast cells, and it can be suggested as a substitute for dental cements exhibiting a long setting time.

  16. Pinus densiflora extract protects human skin fibroblasts against UVB-induced photoaging by inhibiting the expression of MMPs and increasing type I procollagen expression

    Directory of Open Access Journals (Sweden)

    Hoe-Yune Jung

    2014-01-01

    Full Text Available Exposure to ultraviolet (UV light can cause skin photoaging, which is associated with upregulation of matrix metalloproteinases (MMPs and downregulation of collagen synthesis. It has been reported that MMPs, especially MMP-1, MMP-3 and MMP-9, decrease the elasticity of the dermis by degrading collagen. In this study, we assessed the effects of Pinus densiflora extract (PDE on photoaging and investigated its mechanism of action in human skin fibroblast (Hs68 cells after UVB exposure using real-time polymerase chain reaction, Western blot analysis, and enzymatic activity assays. PDE exhibited an antioxidant activity and inhibited elastase activities in vitro. We also found that PDE inhibited UVB-induced cytotoxicity, MMP-1 production and expression of MMP-1, -3 and -9 mRNA in Hs68 cells. In addition, PDE decreased UVB-induced MMP-2 activity and MMP-2 mRNA expression. Moreover, PDE prevented the decrease of type I procollagen mediated by exposure to UVB irradiation, an effect that is linked to the upregulation and downregulation of Smad3 and Smad7, respectively. Another effect of UV irradiation is to stimulate activator protein 1 (AP-1 activity via overexpression of c-Jun/c-Fos, which, in turn, upregulates MMP-1, -3, and -9. In this study, we found that PDE suppressed UV-induced c-Jun and c-Fos mRNA expression. Taken together, these results demonstrate that PDE regulates UVB-induced expression of MMPs and type I procollagen synthesis by inhibiting AP-1 activity and restoring impaired Smad signaling, suggesting that PDE may be useful as an effective anti-photoaging agent.

  17. Low-dose radiation activates Nrf1/2 through reactive species and the Ca(2+)/ERK1/2 signaling pathway in human skin fibroblast cells.

    Science.gov (United States)

    Lee, Eun Kyeong; Kim, Jin-Ah; Park, Seong Joon; Kim, Jeung Ki; Heo, Kyu; Yang, Kwang Mo; Son, Tae Gen

    2013-05-01

    In the current study, we explored the effect of LDR on the activation of Nrfs transcription factor involved in cellular redox events. Experiments were carried out utilizing 0.05 and 0.5 Gy X-ray irradiated normal human skin fibroblast HS27 cells. The results showed LDR induced Nrf1 and Nrf2 activation and expression of antioxidant genes HO-1, Mn-SOD, and NQO1. In particular, 0.05 Gy-irradiation increased only Nrf1 activation, but 0.5 Gy induced both Nrf1 and Nrf2 activation. LDR-mediated Nrf1/2 activation was accompanied by reactive species (RS) generation and Ca(2+) flux. This effect was abolished in the presence of N-acetyl-cysteine and BAPTA- AM. Furthermore, Nrf1/2 activation by LDR was suppressed by PD98059, an inhibitor of ERK1/2. In conclusion, LDR induces Nrf1 and Nrf2 activation and expression of Nrf-regulated antioxidant defense genes through RS and Ca(2+)/ERK1/2 pathways, suggesting new insights into the molecular mechanism underlying the beneficial role of LDR in HS27 cells.

  18. 羊成纤维细胞建系研究进展%The progression of study on goat and sheep fibroblast cell line construction

    Institute of Scientific and Technical Information of China (English)

    豆兴堂

    2014-01-01

    This text has given a summary about goat and sheep fibroblast cell line construction technique and biological characteristics detection, including initial fibroblast cell culture, transfer of culture and purification, cell cryopreservation and resuscitation, testing cell vital force, testing cell growth curve, cell karyotype analysis, et al. The aim was to review achieve-ments of the technique of fibroblast cell line construction, and to look into the future of its us-ing in modern animal husbandry.%本文总结了羊成纤维细胞建系及生物学特性检测技术,包括羊成纤维细胞原代、传代培养与纯化、冷冻保存与复苏、细胞活力测定、生长曲线、染色体分析、同工酶分析、微生物检测等,并回顾了体细胞技术已经取得的成就,展望其在我国现代畜牧业中的应用前景。

  19. Establishment of a transgenic zebrafish line for superficial skin ablation and functional validation of apoptosis modulators in vivo.

    Directory of Open Access Journals (Sweden)

    Chi-Fang Chen

    Full Text Available BACKGROUND: Zebrafish skin is composed of enveloping and basal layers which form a first-line defense system against pathogens. Zebrafish epidermis contains ionocytes and mucous cells that aid secretion of acid/ions or mucous through skin. Previous studies demonstrated that fish skin is extremely sensitive to external stimuli. However, little is known about the molecular mechanisms that modulate skin cell apoptosis in zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: This study aimed to create a platform to conduct conditional skin ablation and determine if it is possible to attenuate apoptotic stimuli by overexpressing potential apoptosis modulating genes in the skin of live animals. A transgenic zebrafish line of Tg(krt4:NTR-hKikGR(cy17 (killer line, which can conditionally trigger apoptosis in superficial skin cells, was first established. When the killer line was incubated with the prodrug metrodinazole, the superficial skin displayed extensive apoptosis as judged by detection of massive TUNEL- and active caspase 3-positive signals. Great reductions in NTR-hKikGR(+ fluorescent signals accompanied epidermal cell apoptosis. This indicated that NTR-hKikGR(+ signal fluorescence can be utilized to evaluate apoptotic events in vivo. After removal of metrodinazole, the skin integrity progressively recovered and NTR-hKikGR(+ fluorescent signals gradually restored. In contrast, either crossing the killer line with testing lines or transiently injecting the killer line with testing vectors that expressed human constitutive active Akt1, mouse constitutive active Stat3, or HPV16 E6 element displayed apoptosis-resistant phenotypes to cytotoxic metrodinazole as judged by the loss of reduction in NTR-hKikGR(+ fluorescent signaling. CONCLUSION/SIGNIFICANCE: The killer/testing line binary system established in the current study demonstrates a nitroreductase/metrodinazole system that can be utilized to conditionally perform skin ablation in a real-time manner, and

  20. Photoprotective Potential of Penta-O-Galloyl-β-DGlucose by Targeting NF-κB and MAPK Signaling in UVB Radiation-Induced Human Dermal Fibroblasts and Mouse Skin.

    Science.gov (United States)

    Kim, Byung-Hak; Choi, Mi Sun; Lee, Hyun Gyu; Lee, Song-Hee; Noh, Kum Hee; Kwon, Sunho; Jeong, Ae Jin; Lee, Haeri; Yi, Eun Hee; Park, Jung Youl; Lee, Jintae; Joo, Eun Young; Ye, Sang-Kyu

    2015-11-01

    Exposure of the skin to ultraviolet radiation can cause skin damage with various pathological changes including inflammation. In the present study, we identified the skin-protective activity of 1,2,3,4,6-penta-O-galloyl-β-D-glucose (pentagalloyl glucose, PGG) in ultraviolet B (UVB) radiation-induced human dermal fibroblasts and mouse skin. PGG exhibited antioxidant activity with regard to intracellular reactive oxygen species (ROS) generation as well as ROS and reactive nitrogen species (RNS) scavenging. Furthermore, PGG exhibited anti-inflammatory activity, inhibiting the activation of nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, resulting in inhibition of the expression of pro-inflammatory mediators. Topical application of PGG followed by chronic exposure to UVB radiation in the dorsal skin of hairless mice resulted in a significant decrease in the progression of inflammatory skin damages, leading to inhibited activation of NF-κB signaling and expression of pro-inflammatory mediators. The present study demonstrated that PGG protected from skin damage induced by UVB radiation, and thus, may be a potential candidate for the prevention of environmental stimuli-induced inflammatory skin damage.

  1. Stimulation of MMP-11 (stromelysin-3 expression in mouse fibroblasts by cytokines, collagen and co-culture with human breast cancer cell lines

    Directory of Open Access Journals (Sweden)

    Matthaei Klaus I

    2004-07-01

    Full Text Available Abstract Background Matrix metalloproteinases (MMPs are central to degradation of the extracellular matrix and basement membrane during both normal and carcinogenic tissue remodeling. MT1-MMP (MMP-14 and stromelysin-3 (MMP-11 are two members of the MMP family of proteolytic enzymes that have been specifically implicated in breast cancer progression. Expressed in stromal fibroblasts adjacent to epithelial tumour cells, the mechanism of MT1-MMP and MMP-11 induction remains unknown. Methods To investigate possible mechanisms of induction, we examined the effects of a number of plausible regulatory agents and treatments that may physiologically influence MMP expression during tumour progression. Thus NIH3T3 and primary mouse embryonic fibroblasts (MEFs were: a treated with the cytokines IL-1β, IL-2, IL-6, IL-8 and TGF-β for 3, 6, 12, 24, and 48 hours; b grown on collagens I, IV and V; c treated with fibronectin, con-A and matrigel; and d co-cultured with a range of HBC (human breast cancer cell lines of varied invasive and metastatic potential. Results Competitive quantitative RT-PCR indicated that MMP-11 expression was stimulated to a level greater than 100%, by 48 hour treatments of IL-1β, IL-2, TGF-β, fibronectin and collagen V. No other substantial changes in expression of MMP-11 or MT1-MMP in either tested fibroblast culture, under any treatment conditions, were observed. Conclusion We have demonstrated significant MMP-11 stimulation in mouse fibroblasts using cytokines, matrix constituents and HBC cell lines, and also some inhibition of MT1-MMP. Our data suggest that the regulation of these genes in the complex stromal-epithelial interactions that occur in human breast carcinoma, is influenced by several mechanisms.

  2. Differential modulation of CXCR4 and CD40 protein levels by skin sensitizers and irritants in the FSDC cell line

    OpenAIRE

    Neves, Bruno Miguel; Cruz, Maria Teresa; Francisco, Vera; Gonçalo, Margarida; Figueiredo, Américo; Duarte, Carlos B.; Lopes, Maria Celeste

    2008-01-01

    The development of non-animal methods for skin sensitization testing is an urgent challenge. Some of the most promising in vitro approaches are based on the analysis of phenotypical and functional modifications induced by sensitizers in dendritic cell models. In this work, we evaluated, for the first time, a fetal skin-derived dendritic cell line (FSDC) as a model to discriminate between sensitizers and irritants, through analysis of their effects on CD40 and CXCR4 protein expression. The che...

  3. Effect of 660 nm Light-Emitting Diode on the Wound Healing in Fibroblast-Like Cell Lines

    Directory of Open Access Journals (Sweden)

    Myung-Sun Kim

    2015-01-01

    Full Text Available Light in the red to near-infrared (NIR range (630–1000 nm, which is generated using low energy laser or light-emitting diode (LED arrays, was reported to have a range of beneficial biological effects in many injury models. NIR via a LED is a well-accepted therapeutic tool for the treatment of infected, ischemic, and hypoxic wounds as well as other soft tissue injuries in humans and animals. This study examined the effects of exposure to 660 nm red LED light at intensities of 2.5, 5.5, and 8.5 mW/cm2 for 5, 10, and 20 min on wound healing and proliferation in fibroblast-like cells, such as L929 mouse fibroblasts and human gingival fibroblasts (HGF-1. A photo illumination-cell culture system was designed to evaluate the cell proliferation and wound healing of fibroblast-like cells exposed to 600 nm LED light. The cell proliferation was evaluated by MTT assay, and a scratched wound assay was performed to assess the rate of migrating cells and the healing effect. Exposure to the 660 nm red LED resulted in an increase in cell proliferation and migration compared to the control, indicating its potential use as a phototherapeutic agent.

  4. Cytotoxicity and Proliferation Studies with Arsenic in Established Human Cell Lines: Keratinocytes, Melanocytes, Dendritic Cells, Dermal Fibroblasts, Microvascular Endothelial Cells, Monocytes and T-Cells

    Directory of Open Access Journals (Sweden)

    Hari H. P. Cohly

    2003-01-01

    Full Text Available Abstract: Based on the hypothesis that arsenic exposure results in toxicity and mitogenecity, this study examined the dose-response of arsenic in established human cell lines of keratinocytes (HaCaT, melanocytes (1675, dendritic cells (THP-1/A23187, dermal fibroblasts (CRL1904, microvascular endothelial cells (HMEC, monocytes (THP-1, and T cells (Jurkat. Cytotoxicity was determined by incubating THP-1, THP-1+ A23187 and JKT cells in RPMI 1640, 1675 in Vitacell, HMEC in EBM, and dermal fibroblasts and HaCaT in DMEM with 10% fetal bovine serum, 1% streptomycin and penicillin for 72 hrs in 96-well microtiter plates, at 37oC in a 5% CO2 incubator with different concentrations of arsenic using fluorescein diacetate (FDA. Cell proliferation in 96-well plates was determined in cultured cells starved by prior incubation for 24 hrs in 1% FBS and exposed for 72 hours, using the 96 cell titer proliferation solution (Promega assay. Cytotoxicity assays yielded LD50s of 9 μg/mL for HaCaT, 1.5 μg/mL for CRL 1675, 1.5 μg/mL for dendritic cells, 37 μg/mL for dermal fibroblasts, 0.48 μg/mL for HMEC, 50 μg/mL for THP-1 cells and 50 μg/mL for JKT-T cells. The peak proliferation was observed at 6 μg/mL for HaCaT and THP-1 cells, 0.19 μg/mL for CRL 1675, dendritic cells, and HMEC, and 1.5 μg/mL for dermal fibroblasts and Jurkat T cells. These results show that arsenic is toxic at high doses to keratinocytes, fibroblasts, monocytes and T cells, and toxic at lower doses to melanocytes, microvascular endothelial cells and dendritic cells. Proliferation studies showed sub-lethal doses of arsenic to be mitogenic.

  5. Laser Doppler line scanner for monitoring skin perfusion changes of port wine stains during vascular-targeted photodynamic therapy

    Science.gov (United States)

    Chen, Defu; Ren, Jie; Wang, Ying; Gu, Ying

    2014-11-01

    Vascular-targeted photodynamic therapy (V-PDT) is known to be an effective therapeutic modality for the treatment of port wine stains (PWS). Monitoring the PWS microvascular response to the V-PDT is crucial for improving the effectiveness of PWS treatment. The objective of this study was to use laser Doppler technique to directly assess the skin perfusion in PWS before and during V-PDT. In this study, 30 patients with PWS were treated with V-PDT. A commercially laser Doppler line scanner (LDLS) was used to record the skin perfusion of PWS immediately before; and at 1, 3, 5, 7, 10, 15 and 20 minutes during V-PDT treatment. Our results showed that there was substantial inter- and intra-patient perfusion heterogeneity in PWS lesion. Before V-PDT, the comparison of skin perfusion in PWS and contralateral healthy control normal skin indicated that PWS skin perfusion could be larger than, or occasionally equivalent to, that of control normal skin. During V-PDT, the skin perfusion in PWS significantly increased after the initiation of V-PDT treatment, then reached a peak within 10 minutes, followed by a slowly decrease to a relatively lower level. Furthermore, the time for reaching peak and the subsequent magnitude of decrease in skin perfusion varied with different patients, as well as different PWS lesion locations. In conclusion, the LDLS system is capable of assessing skin perfusion changes in PWS during V-PDT, and has potential for elucidating the mechanisms of PWS microvascular response to V-PDT.

  6. Dermal Lipogenesis Inhibits Adiponectin Production in Human Dermal Fibroblasts while Exogenous Adiponectin Administration Prevents against UVA-Induced Dermal Matrix Degradation in Human Skin.

    Science.gov (United States)

    Fang, Chien-Liang; Huang, Ling-Hung; Tsai, Hung-Yueh; Chang, Hsin-I

    2016-07-14

    Adiponectin is one of the most abundant adipokines from the subcutaneous fat, and regulates multiple activities through endocrine, paracrine, or autocrine mechanisms. However, its expression in adipogenic induced fibroblasts, and the potential role in photoaging has not been determined. Here, human dermal fibroblasts, Hs68, were presented as a cell model of dermal lipogenesis through stimulation of adipogenic differentiation medium (ADM). Similar to other studies in murine pre-adipocyte models (i.e., 3T3-L1), Hs68 fibroblasts showed a tendency to lipogenesis based on lipid accumulation, triglyceride formation, and the expressions of PPAR-γ, lipoprotein lipase (LPL), and FABP4 mRNA. As expected, ADM-treated fibroblasts displayed a reduction on adiponectin expression. Next, we emphasized the photoprotective effects of adiponectin against UVA-induced damage in Hs68 fibroblasts. UVA radiation can downregulate cell adhesion strength and elastic modulus of Hs68 fibroblasts. Moreover, UVA radiation could induce the mRNA expressions of epidermal growth factor receptor (EGFR), adiponectin receptor 1 (AdipoR1), matrix metalloproteinase-1 (MMP-1), MMP-3, and cyclooxygenase-2 (COX-2), but downregulate the mRNA expressions of type I and type III collagen. On the other hand, post-treatment of adiponectin can partially overcome UVA-induced reduction in the cell adhesion strength of Hs68 fibroblasts through the activation of AdipoR1 and the suppression of EGF-R. In addition, post-treatment of adiponectin indicated the increase of type III collagen and elastin mRNA expression and the decrease of MMP-1 and MMP-3 mRNA expression, but a limited degree of recovery of elastic modulus on UVA-irradiated Hs68 fibroblasts. Overall, these results suggest that dermal lipogenesis may inhibit the expression of adiponectin while exogenous adiponectin administration prevents against UVA-induced dermal matrix degradation in Hs68 fibroblasts.

  7. 脂质体介导的PRX-2转染对人成纤维细胞增殖特性的影响%Effect of PRX-2 gene transferred by lipofactamine on the proliferation of human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    宋慧锋; 柴家科; 林子豪

    2011-01-01

    目的 探讨PRX-2转染对人成纤维细胞增殖特性的影响.方法 取烧伤整形外科手术中所取的全厚皮片的剩余皮片,男5例,女3例.年龄6 ~41岁,平均24岁,均取自腹部.细胞培养采用组织块法,实验用第4代细胞.皮肤组织进行成纤维细胞的培养、传代,利用脂质体介导的基因转染技术,将PRX-2基因转染至人成纤维细胞中,形成稳定表达,利用MTT法、流式细胞仪等方法,观察转染前后人皮肤成纤维细胞增殖特性的变化情况.结果 PRX-2转染后成纤维细胞形态与正常成纤维细胞类似,增殖能力明显增强,G0-G1期细胞比例减少,而S期和G2-M期细胞比例增多.PRX-2基因成功转染到了人成纤维细胞中;细胞增殖指数增高.结论 脂质体介导的同源异形框基因PRX-2转染可以增强人成纤维细胞的增殖能力,PRX-2和人皮肤创面愈合有密切的关系.%Objective To explore the effects of PRX-2 gene transferred by lipofactamine on the human skin fibroblasts.Methods Normal human skin fibroblasts were cultured and PRX-2 gene was transferred by lipofactamine.The proliferation of fibroblasts was detected by MTT (3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) and flow cytometry.Results The proliferation of PRX-2-transfected fibroblasts was stronger than that of normal counterparts.There were fewer cells during G0-G1 period and more cells during S and G2-M periods.The proliferative index increased.Conclusion The proliferation of fibroblasts may be modified by transfected PRX-2.Thus PRX-2 plays an important role during the healing of human skin wound.

  8. Differences between scar and dermal cultured fibroblasts derived from a patient with recurrent abdominal incision wound herniation.

    Science.gov (United States)

    Boemi, L; Allison, G M; Graham, W P; Krummel, T M; Ehrlich, H P

    1999-10-01

    Fibroblasts were derived from dermis and scar of a 47-year-old white man with a recurrent incisional hernia as a result of fractured ribs. The scar was thin and stretched, suggesting a defect in the maturation of granulation tissue. After surgical repair, biopsy specimens of discarded scar and skin were used to generate fibroblast cell lines. Fibroblasts maintained in medium containing 10% fetal bovine serum and antibiotic were studied between their third and eighth passage. By phase contrast microscopy, no structural differences were obvious, but it was noted that to pass scar fibroblasts, a more aggressive trypsin regimen was required. Immunohistologic and Western blot analysis of patient scar fibroblasts showed (1) more a smooth muscle actin within stress fibers, (2) increased expression of the vitronectin integrin receptor alpha(v) (CD 51), and (3) reduced expression of the collagen integrin receptor alpha2 (CD 49b). The expression of vinculin from focal adhesions or a tubulin from microtubules was the same among cell lines. Contractions of scar and dermal fibroblast-populated collagen lattice were compared. At 24 hours, contractions were 69 percent with newborn fibroblasts (normal); 68 percent for patient dermal fibroblasts; and only 48 percent for patient scar fibroblasts. The retarded contraction of scar fibroblast-populated collagen lattice was significant (p > or = 0.002). Myosin ATPase activity, critical for lattice contraction, and cell migration were equivalent among all cell lines. A plausible mechanism for the retardation of scar lattice contraction is disruption of fibroblasts and collagen interactions, for which the attachment of cells to collagen is altered. It is proposed that either the decrease in the expression of collagen integrin receptor alpha2 (CD 49b), an increase in the expression of the vitronectin receptor alpha(v) (CD 51), or a combination of both is responsible for disruption of collagen fibroblast interactions.

  9. Artificial Skin – Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

    OpenAIRE

    Hanna Wendt; Anja Hillmer; Kerstin Reimers; Kuhbier, Joern W.; Franziska Schäfer-Nolte; Christina Allmeling; Cornelia Kasper; Vogt, Peter M.

    2011-01-01

    BACKGROUND: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. METHODOLOGY/PRINCIPAL FINDINGS: Native spider draglin...

  10. Artificial Skin - Culturing of Different Skin Cell Lines for Generating an Artificial Skin Substitute on Cross-Weaved Spider Silk Fibres

    OpenAIRE

    Wendt, Hanna; Hillmer, Anja; REIMERS, KERSTIN; Kuhbier, Joern W.; Schaefer-Nolte, Franziska; Allmeling, Christina; Kasper, Cornelia; Vogt, Peter M.

    2011-01-01

    Background: In the field of Plastic Reconstructive Surgery the development of new innovative matrices for skin repair is in urgent need. The ideal biomaterial should promote attachment, proliferation and growth of cells. Additionally, it should degrade in an appropriate time period without releasing harmful substances, but not exert a pathological immune response. Spider dragline silk from Nephila spp meets these demands to a large extent. Methodology/Principal Findings: Native spider draglin...

  11. Effect of In-Ceram all-ceramic crown and metal-ceramic crowns on cell viability and related gene expression in fibroblast cell lines L929

    Institute of Scientific and Technical Information of China (English)

    Lei Shi; Liang Yang; Jie-Chun Huang; En-Bao He; Zhi-Hui Wu

    2016-01-01

    Objective:To study the effect of In-Ceram all-ceramic crown and metal-ceramic crowns on cell viability and related gene expression in fibroblast cell lines L929.Methods: Fibroblast cell lines L929 were cultured and treated with extract solution of In-Ceram all-ceramic crown, Ni-Cr alloy porcelain crown and Co-Cr alloy porcelain crown respectively, and then cell viability, serum cytokine contents as well as mRNA contents of Fas, FasL, Apo-1, mTOR and P70S6k in cells were detected.Results:Cell OD values of Ni-Cr alloy group and Co-Cr alloy group were significantly lower than that of negative control group; cell OD value of In-Ceram group was significantly higher than those of Ni-Cr alloy group and Co-Cr alloy group; TNF-α, IL-6 and IL-8 contents in cell culture medium of Ni-Cr alloy group and Co-Cr alloy group were significantly higher than those of negative control group, and TNF-α, IL-6 and IL-8 contents in cell culture medium of In-Ceram group were significantly lower than those of Ni-Cr alloy group and Co-Cr alloy group and not different from those of negative control group; mRNA contents of Fas, FasL and Apo-1 in cells of Ni-Cr alloy group and Co-Cr alloy group were higher than those of negative control group, mRNA contents of mTOR and P70S6k were lower than those of negative control group, mRNA contents of Fas, FasL and Apo-1 in cells of In-Ceram group were lower than those of Ni-Cr alloy group and Co-Cr alloy group and not different from those of negative control group, and mRNA contents of mTOR and P70S6k were higher than those of Ni-Cr alloy group and Co-Cr alloy group and not different from those of negative control group.Conclusion: In-Ceram all-ceramic crown has good histocompatibility and will not affect cell viability as well as generation of inflammatory factors and expression of apoptosis and proliferation-related genes in fibroblast cell lines L929.

  12. Acute Activation of Oxidative Pentose Phosphate Pathway as First-Line Response to Oxidative Stress in Human Skin Cells.

    Science.gov (United States)

    Kuehne, Andreas; Emmert, Hila; Soehle, Joern; Winnefeld, Marc; Fischer, Frank; Wenck, Horst; Gallinat, Stefan; Terstegen, Lara; Lucius, Ralph; Hildebrand, Janosch; Zamboni, Nicola

    2015-08-01

    Integrity of human skin is endangered by exposure to UV irradiation and chemical stressors, which can provoke a toxic production of reactive oxygen species (ROS) and oxidative damage. Since oxidation of proteins and metabolites occurs virtually instantaneously, immediate cellular countermeasures are pivotal to mitigate the negative implications of acute oxidative stress. We investigated the short-term metabolic response in human skin fibroblasts and keratinocytes to H2O2 and UV exposure. In time-resolved metabolomics experiments, we observed that within seconds after stress induction, glucose catabolism is routed to the oxidative pentose phosphate pathway (PPP) and nucleotide synthesis independent of previously postulated blocks in glycolysis (i.e., of GAPDH or PKM2). Through ultra-short (13)C labeling experiments, we provide evidence for multiple cycling of carbon backbones in the oxidative PPP, potentially maximizing NADPH reduction. The identified metabolic rerouting in oxidative and non-oxidative PPP has important physiological roles in stabilization of the redox balance and ROS clearance.

  13. Ochratoxin A and T-2 Toxin Induce Clonogenicity and Cell Migration in Human Colon Carcinoma and Fetal Lung Fibroblast Cell Lines.

    Science.gov (United States)

    Abassi, Haila; Ayed-Boussema, Imen; Shirley, Sarah; Abid, Salwa; Bacha, Hassen

    2016-03-01

    T-2 toxin and Ochratoxin A (OTA) are toxic secondary metabolites produced by various fungi, and together they contaminate feedstuffs worldwide. T-2 toxin and OTA may exert carcinogenic action in rodent. Despite the various in vivo experiments, carcinogenicity of these two mycotoxins has not yet been proven for human. In this current study, we proposed to investigate, in Human colon carcinoma cells and fetal lung fibroblast-like cells transfected with MYC, the effect of T-2 toxin and OTA on cell clonogenicity and cell migration. Results of the present investigation showed that T2-toxin as well as OTA has an important clonogenic effect in all cell lines, suggesting that these mycotoxins could promote the transcription of c-myc gene. Furthermore, T-2 toxin and OTA enhanced the migration effect of HCT116 cells at very low concentrations, proposing that these mycotoxins may exhibit carcinogenesis-like properties in the studied cells.

  14. Chitosan/TiO2 composite membrane improves proliferation and survival of L929 fibroblast cells: Application in wound dressing and skin regeneration.

    Science.gov (United States)

    Behera, Sudhanshu Shekhar; Das, Urmimala; Kumar, Awanish; Bissoyi, Akalabya; Singh, Abhishek Kumar

    2017-05-01

    The microbial infection and growth of fibroblasts are the critical factors for the effective wound healing. The natural polymer-based dressing membranes may mimic extracellular matrix to support the survival, proliferation and differentiation of fibroblasts. The present study deals with the preparation of chitosan/titanium dioxide (CS/TiO2) composite membranes with different degree of TiO2 incorporation, and their characterization in terms of morphology, ultrastructure, thermal behavior and mechanical properties with SEM, FTIR, XRD and tensile strength analyses. The data demonstrated the formation of strong O-Ti-O bonding between TiO2 and CS resulting in superior porosity, mechanical strength, crystallinity and flexibility of the composite membranes. Further, the cyto-compatibility, proliferation, oxidative stress, cell cycle and apoptosis analyses of fibroblast L929 cells demonstrated the enhanced proliferation and survival, and decreased oxidative stress and apoptosis in L929 cells grown on CS/TiO2 membrane incorporated with 025% TiO2. Next, we measured the significant up-regulation in the expression of fibroblast-markers in L929 cells cultured on CS/TiO2 (0.25%) membrane. Furthermore, the CS/TiO2 composite membranes exhibited a superior antibacterial activity against Staphylococcus aureus. Taken together, the data confirmed that CS/TiO2 (0.25%) membrane improved the growth, survival and functional integrity of fibroblasts, and exerted antibacterial activity which may be utilized as potential dressing materials.

  15. Epigenetic and phenotypic profile of fibroblasts derived from induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Kyle J Hewitt

    Full Text Available Human induced pluripotent stem (hiPS cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK and their hES-derived counterparts (EDK showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM-production (COL1A1 by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ, revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application.

  16. 脂多糖干预后人皮肤成纤维细胞的胶原代谢%Influence of Lipopolysaccharide on Collagen Metabolism of Normal Skin Fibroblasts of Human

    Institute of Scientific and Technical Information of China (English)

    李凤玉; 王舒琦; 贾国洪; 万立; 杨红明

    2012-01-01

    目的 探讨脂多糖(lipopolysaccharide,LPS)对皮肤成纤维细胞胶原代谢的影响,以了解LPS在增生性瘢痕形成中的生物学作用.方法 取正常皮肤行成纤维细胞培养后,分为1个对照组及6个实验组.实验组终浓度分别为0.005、0.010、0.050、0.100、0.500和1.000 μg/ml大肠杆菌LPS(E.coli055:B5)培养,对照组为DMEM培养.用逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)测定成纤维细胞Ⅰ、Ⅲ型前胶原mRNA及胶原酶mRNA的表达,并以同一个体相同代数的瘢痕组织成纤维细胞做对照.结果 与对照组比较,0.005~0.500μg/ml组促进细胞胶原合成,且0.100 μg/ml组作用达高峰(P<0.05),1.000μg/ml LPS抑制细胞胶原合成(P≮0.05).LPS刺激浓度在0.005 μg/ml~0.100μg/ml时,促进正常皮肤成纤维细胞I、Ⅲ型前胶原mRNA表达,抑制胶原酶mRNA表达,且呈一定的剂量依赖性;当LPS刺激浓度为0.500 μg/ml,上述作用下降;而当LPS刺激浓度到达1.000 μg/ml时,抑制正常皮肤成纤维细胞Ⅰ、Ⅲ型前胶原mRNA表达,促进胶原酶mRNA表达.LPS刺激浓度在0.100 μg/ml时,成纤维细胞Ⅰ、Ⅲ型前胶原mRNA和胶原酶mRNA表达与同一个体增生性瘢痕组织成纤维细胞近似.结论 LPS对人皮肤成纤维细胞Ⅰ、Ⅲ型前胶原mRNA和胶原酶mRNA表达的直接调节,可能是其参与增生性瘢痕形成的重要机制.%Objective To observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar. Methods Fibroblasts were isolated and cultured in vitro, and then exposed to different doses of LPS (0. 005, 0. 010, 0. 050, 0. 100, 0. 500 and 1. 000 jug/ml) from E. coli055: B5 respectively. The expression of procollagen type I , HI and collagenase mRNAs was tested by reverse transcription-polymerase chain reaction (RT-PCR). The fibroblasts from hypertrophic scar tissue

  17. Inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts%绿原酸抗人皮肤成纤维细胞衰老的研究

    Institute of Scientific and Technical Information of China (English)

    陈婷; 江智茂; 于波; 马刚

    2015-01-01

    [Abstract ] Objective To evaluate the inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts (HSFs). Methods Fibroblasts isolated from human foreskin were treated with 1 mmol/L glyoxal in vitro to develop a model for cellular senescence. In order to select effective concentrations of chlorogenic acid, some HSFs were treated with 1 mmol/L glyoxal alone or in combination with chlorogenic acid at different concentrations (5, 10, 20, 40, 80 μmol/L)for 3 days, with those receiving no treatment serving as the blank control group. Then, methyl thiazolyl tetrazolium (MTT)assay was performed to evaluate the proliferative activity of HSFs. Some HSFs were divided into 5 groups to be cultured alone(blank control group), or treated with 1 mmol/L glyoxal(glyoxal group)or the combination of 1 mmol/L glyoxal and chlorogenic acid at effective concentrations of 10, 20 and 40 μmol/L (glyoxal + chlorogenic acid groups). Senescence associated β-galactosidase (SA-β-gal)staining and real-time fluorescence-based quantitative PCR were conducted to determine the percentage of senescent cells and expression level of p16INK4a mRNA respectively. Statistical analysis was carried out by one-way analysis of variance followed by the least significant difference(LSD)-t test. Results Compared with the blank control group, the glyoxal group showed significantly decreased cellular proliferative activity of HSFs (55.65% ± 2.00% vs. 100% ± 6.90%, P 0.05). Therefore, 10 - 40 μmol/L was selected as the effective concentrations of chlorogenic acid. The glyoxal group showed significant increases in the percentage of senescent (SA-β-gal-positive)cells (35.65% ± 2.24% vs. 13.00% ± 2.22%, P 0.05),因此筛选出绿原酸的有效浓度为10~40μmol/L。与空白对照组衰老细胞比例(13.00%±2.22%)比较,加入乙二醛后,衰老细胞明显增多(35.65%±2.24%),差异有统计学意义(P <0.01)。与乙二醛组细胞 SA-β-gal 染色

  18. Cytotoxic Evaluation of Elastomeric Dental Impression Materials on a Permanent Mouse Cell Line and on a Primary Human Gingival Fibroblast Culture

    Directory of Open Access Journals (Sweden)

    Roberta Tiozzo

    2009-08-01

    Full Text Available The need for clinically relevant in vitro tests of dental materials is widely recognized. Nearly all dental impression materials are introduced into the mouth just after mixing and allowed to set in contact with the oral tissues. Under these conditions, the materials may be toxic to cells or may sensitize the tissues. The aim of the present study is to evaluate the potential cytotoxicity of new preparations of elastomeric dental impression materials: A four vinylpolysiloxanes: Elite H-D Putty and Elite H-D Light Body (Zhermack, Badia Polesine, Rovigo, Italy; Express Putty and Express Light Body (3M ESPE AG Seefeld, Germany and B two polyethers: Impregum Penta and Permadyne Penta L (3M ESPE AG Seefeld, Germany. The cytotoxicity of these impression materials were examined using two different cell lines: Balb/c 3T3 (permanent cell line and human gingival fibroblasts (primary cell line and their effects were studied by indirect and direct tests. The direct tests are performed by placing one sample of the impression materials in the centre of the Petri dishes at the time of the seeding of cells. The cell growth was evaluated at the 12th and 24th hours by cell number. The indirect tests were performed by incubating a square of 1 cm diameter impression material in 5 mL of medium at 37 °C for 24 hours (“eluates”. Subconfluent cultures are incubated with “eluates” for 24 hours. The MTT-formazan production is the method used for measuring the cell viability. The results indicate that: a polyether materials are cytotoxic under both experimental conditions; b among vinylpolysiloxanes, only Express Light Body (3M ESPE AG Seefeld, Germany induces clear inhibition of cellular viability of Balb/c 3T3 evaluated by direct and indirect tests and c the primary cell line is less sensitive to the toxic effect than the permanent cell line.

  19. Characteristic Gene Expression Profiles of Human Fibroblasts and Breast Cancer Cells in a Newly Developed Bilateral Coculture System

    Directory of Open Access Journals (Sweden)

    Takayuki Ueno

    2015-01-01

    Full Text Available The microenvironment of cancer cells has been implicated in cancer development and progression. Cancer-associated fibroblast constitutes a major stromal component of the microenvironment. To analyze interaction between cancer cells and fibroblasts, we have developed a new bilateral coculture system using a two-sided microporous collagen membrane. Human normal skin fibroblasts were cocultured with three different human breast cancer cell lines: MCF-7, SK-BR-3, and HCC1937. After coculture, mRNA was extracted separately from cancer cells and fibroblasts and applied to transcriptomic analysis with microarray. Top 500 commonly up- or downregulated genes were characterized by enrichment functional analysis using MetaCore Functional Analysis. Most of the genes upregulated in cancer cells were downregulated in fibroblasts while most of the genes downregulated in cancer cells were upregulated in fibroblasts, indicating that changing patterns of mRNA expression were reciprocal between cancer cells and fibroblasts. In coculture, breast cancer cells commonly increased genes related to mitotic response and TCA pathway while fibroblasts increased genes related to carbohydrate metabolism including glycolysis, glycogenesis, and glucose transport, indicating that fibroblasts support cancer cell proliferation by supplying energy sources. We propose that the bilateral coculture system using collagen membrane is useful to study interactions between cancer cells and stromal cells by mimicking in vivo tumor microenvironment.

  20. Increased transcription of the c-myc oncogene in two methylcholanthrene-induced quail fibroblastic cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Saule, S.; Martin, P.; Gegonne, A.; Begue, A.; Lagrou, C.; Stehelin, D.

    1984-12-01

    The expression of three c-onc genes (c-erb, c-myc, c-myb) was investigated in five cell lines established from fibrosarcomas induced with 20-methylcholanthrene (MCA) of Japanese quails. These cell lines showed low levels of the three c-onc genes, with the exception of two cell lines that accumulated moderate (MCAQ 1-4) and large amounts (MCAQ 3-5) of c-myc RNA. Molecular cloning and restriction endonuclease analyses indicated that expression of c-myc in these two cell lines were not associated with detectable rearrangements in the c-myc locus, that the size of the c-myc transcript (2.7 kb) in MCAQ 3-5 was similar to that of the normal c-myc messenger RNAs (mRNA) and that the transcriptional activatin observed in MCAQ 3-5 was not mediated by the LTR (long terminal repeat) of a proximate ALV (avian leukosis virus) provirus. Finally, when analyzed with the restriction enzymes Msp I and Hpa II, the c-myc locus of MCAQ 3-5 and MCAQ 1-4 was found hypomethylated as compared with that of the other cell lines tested that show low levels of c-myc transcripts. Results suggest that one of the ways methylcholanthrene could mediate transformation is by inducing an abnormal regulation of the c-myc gene.

  1. Photoprotection of Skin Fibroblasts from Uitraviolet Radiation by Notoginsenoside R1%三七皂苷R1对UV辐射皮肤成纤维细胞的影响

    Institute of Scientific and Technical Information of China (English)

    谢璟; 何黎; 郝萍; 万屏

    2011-01-01

    Objective To observe the photoprotection of notoginsenoside R1 on fibroblasts after UVB and UVA irradiation and to explore the relative molecular mechanism. Methods Fibroblasts were obtained from human skin. The cells were irradiated with different dosages of UV and treated with notoginsenoside R1. The damage of fibroblasts was observed with inverted phase contrast microscope. Cellular proliferation activity was detected by MTT method. The hy-droxyproline amount in the culture supernatant was measured by colorimetric method, and the level of matrix metal-loproteinase-1 (MMP-1) secreted by fibroblasts was measured with ELISA kits. Results The irradiation damage degree of the cells was dependent with the irradiated dose. The number and morphology of the cells remained normal when they were pretreated with notoginsenoside R1. The activity of proliferation of fibroblasts irradiated by UV was increased and MMP-1 protein secretion level was suppressed when the dose of notoginsenoside R1 was 5 μg· mL-1 and 20 μg· mL-1. Conclusion UVA and UVB irradiation can induce obvious damage to fibroblasts of human skin. Notoginsenoside R1 protects the damage of fibroblasts from UV irradiation.%目的 探讨三七皂苷R1对UV诱导的人皮肤成纤维细胞的保护作用及其相关分子机制.方法 采用不同剂量UV照射原代培养的人皮肤成纤维细胞,同时加入三七皂苷R1干预处理,倒置相差显微镜观察细胞损伤的形态学变化,并用MTT法检测其增殖活性,比色法检测成纤维细胞培养上清中羟脯氨酸的含量,ELISA法检测成纤维细胞MMP-1蛋白水平.结果 UVB、UVA辐射成纤维细胞后,细胞损伤程度呈剂量依赖性,加入三七皂苷R1后,体外培养成纤维细胞未出现明显的数量上和形态学改变;三七皂苷R1浓度为5,20 μg·mL-1时经UV照射的FB细胞增殖活性增加;三七皂苷R1浓度为5,20 μg· mL-1时抑制MMP-1的分泌.结论 UVB、UVA辐射对皮肤成纤维细胞具有明

  2. Whole-exome sequencing of fibroblast and its iPS cell lines derived from a patient diagnosed with xeroderma pigmentosum

    Directory of Open Access Journals (Sweden)

    Kohji Okamura

    2015-12-01

    Full Text Available Cells from a patient with a DNA repair-deficiency disorder are anticipated to bear a large number of somatic mutations. Because such mutations occur independently in each cell, there is a high degree of mosaicism in patients' tissues. While major mutations that have been expanded in many cognate cells are readily detected by sequencing, minor ones are overlaid with a large depth of non-mutated alleles and are not detected. However, cell cloning enables us to observe such cryptic mutations as well as major mutations. In the present study, we focused on a fibroblastic cell line that is derived from a patient diagnosed with xeroderma pigmentosum (XP, which is an autosomal recessive disorder caused by a deficiency in nucleotide excision repair. By making a list of somatic mutations, we can expect to see a characteristic pattern of mutations caused by the hereditary disorder. We cloned a cell by generating an iPS cell line and performed a whole-exome sequencing analysis of the progenitor and its iPS cell lines. Unexpectedly, we failed to find causal mutations in the XP-related genes, but we identified many other mutations including homozygous deletion of GSTM1 and GSTT1. In addition, we found that the long arm of chromosome 9 formed uniparental disomy in the iPS cell line, which was also confirmed by a structural mutation analysis using a SNP array. Type and number of somatic mutations were different from those observed in XP patients. Taken together, we conclude that the patient might be affected by a different type of the disorder and that some of the mutations that we identified here may be responsible for exhibiting the phenotype. Sequencing and SNP-array data have been submitted to SRA and GEO under accession numbers SRP059858 and GSE55520, respectively.

  3. Mouse fibroblast L929 cells are less permissive to infection by Nelson Bay orthoreovirus compared to other mammalian cell lines.

    Science.gov (United States)

    Mok, Lawrence; Wynne, James W; Grimley, Samantha; Shiell, Brian; Green, Diane; Monaghan, Paul; Pallister, Jackie; Bacic, Antony; Michalski, Wojtek P

    2015-07-01

    In recent years, bats have been identified as a natural reservoir for a diverse range of viruses. Nelson Bay orthoreovirus (NBV) was first isolated from the heart blood of a fruit bat (Pteropus poliocephalus) in 1968. While the pathogenesis of NBV remains unknown, other related members of this group have caused acute respiratory disease in humans. Thus the potential for NBV to impact human health appears plausible. Here, to increase our knowledge of NBV, we examined the replication and infectivity of NBV using different mammalian cell lines derived from bat, human, mouse and monkey. All cell lines supported the replication of NBV; however, L929 cells showed a greater than 2 log reduction in virus titre compared with the other cell lines. Furthermore, NBV did not induce major cytopathic effects in the L929 cells, as was observed in other cell lines. Interestingly, the related Pteropine orthoreoviruses, Pulau virus (PulV) and Melaka virus (MelV) were able to replicate to high titres in L929 cells but infection resulted in reduced cytopathic effect. Our study demonstrates a unique virus-host interaction between NBV and L929 cells, where cells effectively control viral infection/replication and limit the formation of syncytia. By elucidating the molecular mechanisms that control this unique relationship, important insights will be made into the biology of this fusogenic virus.

  4. 无机砷对人皮肤成纤维细胞缝隙连接通讯的影响%Effect of inorganic arsenic on gap junctional intercellular communication between human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    邓芙蓉; 郭新彪

    2001-01-01

    目的探讨无机砷对人皮肤成纤维细胞缝隙连接通讯的影响。方法采用细胞划痕染料标记示踪技术,以荧光染料在细胞间的扩散作为评价缝隙连接通讯的指标,观察亚砷酸和砷酸对原代培养的人皮肤成纤维细胞缝隙连接通讯的影响。结果亚砷酸可显著抑制人皮肤成纤维细胞间荧光黄染料的扩散,在0.005~5.0 μmol/L浓度范围内有明显的剂量依赖关系。砷酸也可明显抑制荧光黄染料在人皮肤成纤维细胞间的扩散,但剂量依赖关系不明显。进一步的研究发现,亚砷酸可显著增高人皮肤成纤维细胞胞膜和胞浆蛋白激酶C的活性,其中对胞膜蛋白激酶C活性的作用更为明显。结论无机砷可显著抑制人皮肤成纤维细胞缝隙连接通讯,提示它们在癌症发生的促长阶段扮演重要角色。无机砷对细胞缝隙连接通讯的抑制可能与其引起细胞内蛋白激酶C的异常活化有关。%Objective The aim of this study is to investigate the effect of inorganic arsenic on gap junctional intercellular communication (GJIC) between human skin fibroblasts.Methods GJIC between human skin fibroblasts was detected by the scrape loading/dye transfer assay.Results Arsenite at concentrations from 0.005 μmol/L to 5.0 μmol/L significantly inhibited the dye transfer between human skin fibroblasts in a dose dependent manner.The dye transfer between human skin fibroblasts was also inhibited by arsenate,while the dose-dependent effects were not observed.Further study found that arsenite at concentrations of 0.5 μmol/L and 5.0 μmol/L significantly increased protein kinase C (PKC) activities in cells,especially in the membrane fraction.Conclusions Arsenite and arsenate significantly inhibited GJIC between human skin fibroblasts,suggesting that inorganic arsenic may play an important role in the process of tumor promotion.Furthermore,PKC activation may be involved in the

  5. Study of primary culture of human skin scar fibroblasts and their growth curve%人皮肤瘢痕成纤维细胞原代培养及其生长曲线的研究

    Institute of Scientific and Technical Information of China (English)

    沈才齐; 金培生; 李雪阳; 张爱君; 陶常波

    2011-01-01

    目的 建立可靠的人皮肤瘢痕成纤维细胞培养方法,探索细胞的生长规律,为皮肤瘢痕体外研究提供平台.方法 采用组织块贴壁法原代培养瘢痕成纤维细胞,波形蛋白免疫细胞化学法鉴定成纤维细胞,CCK-8测定细胞生长曲线.结果 人皮肤瘢痕原代成纤维细胞培养成功,细胞形态为长梭形,波形蛋白表达阳性,16~18天可传第1代,以后每7~8天可传1代,传代后5~6天时为活跃期,第7天进入停滞期.结论 培养出的人皮肤瘢痕成纤维细胞在传代后第5~6天最适于于体外实验研究.%Objective To establish a reliable cultivation method for human skin scar fihroblasts and explore the growth pattern of the cells, and provide a plat for in vitro research of human skin scars. Methods Primary culture of human skin scar fibrohlasts was performed with routine tissue hlock adhering to the plate wall, and the fibrohlasts were identified by immunocytochemical staining of the vimentin, and cell growth curve was measured by CCK - 8. Results The primary culture of skin scar fibroblasts was successful. The fihroblasts have long - spindle shapes in cell morphology and positive vimentin expression. It takes 16 to 18 days for the first generation, and 7 to 8 days is required for each subculture. From the 5th to the 6th day after subculture. the cells were in the active period, while on the 7th day , the cells entered the period of stagnation. Conclusion Human skin scar fibroblasts can be cultured in stable condition in vitro. and the period from the 5th to 6th day after subculture is the most adequate time for in vitro experiments.

  6. Protective Effect of Boric Acid on Oxidative DNA Damage In Chinese Hamster Lung Fibroblast V79 Cell Lines

    Directory of Open Access Journals (Sweden)

    SezenYılmaz

    2016-02-01

    Full Text Available Objective: Many studies have been published on the antioxidative effects of boric acid (BA and sodium borates in in vitro studies. However, the boron (B concentrations tested in these in vitro studies have not been selected by taking into account the realistic blood B concentrations in humans due to the lack of comprehensive epidemiological studies. The recently published epidemiological studies on B exposure conducted in China and Turkey provided blood B concentrations for both humans in daily life and workers under extreme exposure conditions in occupational setting. The results of these studies have made it possible to test antioxidative effects of BA in in vitro studies within the concentration range relevant to humans. The aim of this study was to investigate the protective effects of BA against oxidative DNA damage in V79 (Chinese hamster lung fibroblast cells. The concentrations of BA tested for its protective effect was selected by taking the blood B concentrations into account reported in previously published epidemiological studies. Therefore, the concentrations of BA tested in this study represent the exposure levels for humans in both daily life and occupational settings. Materials and Methods: In this experimental study, comet assay and neutral red uptake (NRU assay methods were used to determinacy to toxicity and genotoxicity of BA and hydrogen peroxide (H2O2. Results: The results of the NRU assay showed that BA was not cytotoxic within the tested concentrations (3, 10, 30, 100 and 200 μM. These non-cytotoxic concentrations were used for comet assay. BA pre-treatment significantly reduced (P<0.05, one-way ANOVA the DNA damaging capacity of H2O2 at each tested BA concentrations in V79 cells. Conclusion: Consequently, pre-incubation of V79 cells with BA has significantly reduced the H2O2-induced oxidative DNA damage in V79 cells. The protective effect of BA against oxidative DNA damage in V79 cells at 5, 10, 50, 100 and 200 μM (54

  7. Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury

    OpenAIRE

    Jimenez, Ana Gabriela; Harper, James M.; Simon A. Queenborough; Williams, Joseph B.

    2013-01-01

    A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cel...

  8. Differences in gene expressions between synovioblast and skin fibroblast in patients with osteoarthritis%骨关节炎滑膜成纤维细胞与皮肤成纤维细胞基因表达的差异

    Institute of Scientific and Technical Information of China (English)

    吕猛; 肖德明; 杨述华; 林博文; 徐忠世; 陈蓟; 王巨; 丑莉莉

    2007-01-01

    BACKGROUND: During recent years, mononucleotide polymorphism of some genes is possibly related to affectability of osteoarthritis (OA). However, previous researches mainly compare the gene expression of synoviocytes between OA and rheumatoid arthritis (RhA); therefore, the correlation of gene expression between synovioblast and fibroblast in other tissues should be further studied as compared with OA.OBJ ECTIVE: To observe the differences of gene expression between OA synovioblast and skin fibroblast.DESIGN: Observational contrast analysis.SETTING: People's Hospital of Shenzhen City.PARTICIPANTS: Synovium tissue was derived from OA patients who received replacement of knee joint in the Department of Orthopaedics, People's Hospital of Shenzhen City. All OA patients met the diagnostic criteria of osteoarthritis established by American College of Rheumatology in 1995. Three patients including 1 male and 2 females aged more than 65 years old and they did not have cardiac and pulmonary disease and diabetes mellitus. Three male normal volunteers who aged 25 to 35 years did not have rheumatic disease, osteoarthritis and dermatosis. All subjects provided a confirmed consent. The main reagents were detailed as follows: RPMI1640 culture medium, fetal bovine serum and TRIZOL agent (Invitrogen Life Technologies Company, USA); pGEM-T pUC (Progema Company, USA);Display PROFILE-BASIC and Display PROFILE Probe kits (Qbiogen Company, USA).METHODS: The experiment was carried out in People's Hospital of Shenzhen City from January to June 2005. Synovium of OA patients were treated with primary culture to obtain synovioblast; meanwhile, skin fibroblast treated with primary culture from normal subjects was regarded as the control group. Restricted enzyme section differential display was used to separate the different-expressed genes of synovioblast and skin fibroblast in OA patients. In addition, blast technique was used to compare the resulted ranks with Genbank ranks.MAIN OUTCOME

  9. Characterization of a novel fibroblast growth factor 10 (Fgf10 knock-in mouse line to target mesenchymal progenitors during embryonic development.

    Directory of Open Access Journals (Sweden)

    Elie El Agha

    Full Text Available Fibroblast growth factor 10 (Fgf10 is a key regulator of diverse organogenetic programs during mouse development, particularly branching morphogenesis. Fgf10-null mice suffer from lung and limb agenesis as well as cecal and colonic atresia and are thus not viable. To date, the Mlcv1v-nLacZ-24 transgenic mouse strain (referred to as Fgf10(LacZ, which carries a LacZ insertion 114 kb upstream of exon 1 of Fgf10 gene, has been the only strain to allow transient lineage tracing of Fgf10-positive cells. Here, we describe a novel Fgf10(Cre-ERT2 knock-in line (Fgf10(iCre in which a Cre-ERT2-IRES-YFP cassette has been introduced in frame with the ATG of exon 1 of Fgf10 gene. Our studies show that Cre-ERT2 insertion disrupts Fgf10 function. However, administration of tamoxifen to Fgf10(iCre; Tomato(flox double transgenic embryos or adult mice results in specific labeling of Fgf10-positive cells, which can be lineage-traced temporally and spatially. Moreover, we show that the Fgf10(iCre line can be used for conditional gene inactivation in an inducible fashion during early developmental stages. We also provide evidence that transcription factors located in the first intron of Fgf10 gene are critical for maintaining Fgf10 expression over time. Thus, the Fgf10(iCre line should serve as a powerful tool to explore the functions of Fgf10 in a controlled and stage-specific manner.

  10. 补益营卫方、TGFβ1及bFGF对衰老人皮肤成纤维细胞c-Jun基因表达的影响%c-Jun Gene Expression Of The Recipe Of Supplementing Ying-wei And TGF-β1 And bFGF In the aging Human Skin Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    周小平; 司晓伟; 徐武清; 俞洋

    2015-01-01

    进c-Jun基因表达存在不依赖于TGF-β1和bFGF的途径.bFGF蛋白是TGF-β1信号调节途径的下游蛋白;是c-Jun基因表达的重要促进因子.%Objective To study the c-Jun gene expression influenced by the recipe of supplementing Ying-wei and transforming growth factor-β1(TGF-β1) and basic fibroblast growth factor(bFGF) in the aging human skin fibroblasts. Methods Firstly,to copy the aging model of human skin fibroblasts by continuous passage culturing.Secondly,to detect the c-Jun gene expression of the aging human skin fibroblasts through the fluorescent quantitative PCR method,after neutralizing antibody blocking TGF-β1 and bFGF. Results When the human skin fibroblasts aging,the expressive level of c-Jun gene,compared to that of young skin fibroblasts,falls by 70%and 71%in 24h and 48h.But theexpressive levels in 72h are both similar.As aging human skin fibroblasts treated by the recipe of supplementing Ying-wei,the c-Jun gene expression which has increased to 4.67 times and 2.12 times in 24h and 48h,has significantly improved,compared to the aging human skin fibroblasts.And the expressive levels of them are equivalent in 72h.After neutralizing TGF-β1 protein,c-Jun gene expressive level of the aging human skin fibroblasts is 2.4 times and 1.7 times as large as that of young skin fibroblasts in 24h and 72h.In 48h,the gene expressive level of young skin fibroblasts is 9.2 times as large as that of aging human skin fibroblasts.After using the recipe of supplementing Ying-wei,the c-Jun gene expression of aging human skin fibroblasts has improved obviously.The c-Jun gene expressive level of human skin fibroblasts raise by 2.3 times and 1.5 times in 48h and 72h.After neutralizing TGF-β1 protein and bFGF protein,c-Jun gene expressive level is decreased obviously with different time;Compared with the young skin fibroblasts,c-Jun gene expression of the aging human skin fibroblasts falls by 64.5%,94.2%and 73.7%in 24h,48h and 72h.As aging human skin

  11. Aging skin.

    Science.gov (United States)

    Bolognia, J L

    1995-01-16

    Aging of the skin is a composite of actinic damage, chronologic aging, and hormonal influences. The majority of changes associated with aging, such as wrinkles and solar lentigines ("liver spots"), are due to photoaging and reflect cumulative sun exposure as well as skin pigmentation. Classically, chronologic aging includes those cutaneous changes that occur in non-sun-exposed areas, such as the buttocks, and are observed in both men and women. A clinical example would be soft tissue sagging due to elastic fiber degeneration. In women, investigations into the effect of hormones on aging of the skin have concentrated on estrogens; in men, there have been a limited number of studies on the influence of testosterone. The latter have shown an age-dependent decrease in tissue androgens in pubic skin, but not scrotal or thigh skin. To date, age has not been shown to have an effect on androgen receptor binding, although a decrease in foreskin 5 alpha-reductase activity with increasing age has been described. In fibroblast cultures from foreskins, there have been conflicting results as to whether 5 alpha-reductase activity decreases in an age-dependent manner. Some of the skin changes that have been categorized as secondary to chronologic aging, such as decreased sebaceous gland activity and decreased hair growth, may actually represent a decline in the concentration of tissue androgens with increasing age. The influence of androgens on age-related changes in keratinocyte and fibroblast function remains speculative.

  12. Characteristics of changes in the number of yH2AX and Rad51 protein foci in human skin fibroblasts after prolonged exposure to low-dose rate X-ray radiation

    Directory of Open Access Journals (Sweden)

    Ozerov I.V.

    2014-12-01

    Full Text Available Aim: to compare the repair process of DNA double-strand breaks in mammalian cells after acute versus prolonged exposure to X-ray irradiation with different dose rates. Material and methods. Studies were performed on primary human fibroblasts isolated from skin biopsies of healthy volunteers (women, 29 and 30 years. Cells were irradiated using an X-ray machine RUB RUST-M1 (JSC "Ruselectronics", Moscow, Russia at 37°C temperature with a dose rate of 400 mGy/min (200 kV, 2*2.4 mA, a filter of 1.5mm AI or 4 mGy/min (50 kV, 2*0.4 mA, a filter of 1.5 mm AI. Immuno-cytochemical protein staining was utilized for yH2AX and Rad51 foci analysis. Results. Phosphorylated histone H2AX (yH2AX and the key protein of homologous recombination Rad51 foci formation and disappearance kinetics were investigated simultaneously in primary human dermal fibroblasts after acute and prolonged exposure to X-ray radiation at a same dose. It was shown that the relative yield of yH2AX foci per dose reduces with decrease in dose rate, while the relative yield of Rad51 foci conversely increases. Conclusion. Our findings suggest the fundamental differences in the ratio of non-homologous end joining and homologous recombination DNA repair in acute versus prolonged irradiated cells.

  13. Effects of basic fibroblast growth factor on expressions of collagen and fibronectin in normal skin and hypertrophic scar fibroblasts%碱性成纤维细胞生长因子对增生性瘢痕与正常皮肤成纤维细胞胶原、纤维连接蛋白表达的影响

    Institute of Scientific and Technical Information of China (English)

    宋瑞; 卞徽宁; 赖文; 陈德华; 赵克森

    2010-01-01

    背景:碱性成纤维细胞生长因子能促进愈合伤口产生胶原蛋白、纤维连接蛋白和基质酶的基质成分.然而,细胞增殖、细胞外基质及新生血管的形成或伤口基质重塑过程失调,会导致瘢痕组织过度增殖.目的:观察碱性成纤维细胞生长因子在正常皮肤创面愈合和增生性瘢痕形成中的作用.方法:从5例进行瘢痕修复手术患者身上同时取正常皮肤和增生性瘢痕组织,分离培养正常人皮肤成纤维细胞和增生性瘢痕成纤维细胞.应用RT-PCR和酶联免疫吸附法检测两种成纤维细胞胶原、纤维连接蛋白基因表达和蛋白合成.采用JC-1染色和流式细胞术测定成纤维细胞线粒体膜电位改变,采用化学发光法检测细胞内ATP水平改变.观察碱性成纤维细胞生长因子对两种细胞的上述指标的影响.结果与结论:不同浓度碱性成纤维细胞生长因子可减慢增生性瘢痕成纤维细胞生长,抑制增生性瘢痕成纤维细胞Ⅰ型胶原表达和合成(P<0.05).碱性成纤维细胞生长因子对正常皮肤和增生性瘢痕成纤维细胞Ⅲ型胶原表达和合成均无影响.然而可上调正常皮肤成纤维细胞表达纤维连接蛋白(P<0.05).此外,10,100 μg/L碱性成纤维细胞生长因子处理后增生性瘢痕成纤维细胞线粒体膜电位呈去极化趋势,正常皮肤成纤维细胞中ATP水平显著增高(P<0.05).结果表明,碱性成纤维细胞生长因子在正常皮肤创面愈合和增生性瘢痕形成中可能有不同的作用和机制.%BACKGROUND:Basic fibroblast growth factor(bFGF)can promote production of collagen,fibronectin and matrix enzyme in healing wounds.However,dysregulation of this process,such as the abnormal coordination of cell proliferation,extracellular.matrix and neovasculadzation formation,or remodeling of the wound matrix will lead to excess accumulation of scar tissues.OBJECTIVE:To investigate effects of bFGF on normal skin wound healing and

  14. Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells.

    Science.gov (United States)

    Abyzov, Alexej; Mariani, Jessica; Palejev, Dean; Zhang, Ying; Haney, Michael Seamus; Tomasini, Livia; Ferrandino, Anthony F; Rosenberg Belmaker, Lior A; Szekely, Anna; Wilson, Michael; Kocabas, Arif; Calixto, Nathaniel E; Grigorenko, Elena L; Huttner, Anita; Chawarska, Katarzyna; Weissman, Sherman; Urban, Alexander Eckehart; Gerstein, Mark; Vaccarino, Flora M

    2012-12-20

    Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) has been suspected of causing de novo copy number variation. To explore this issue, here we perform a whole-genome and transcriptome analysis of 20 human iPSC lines derived from the primary skin fibroblasts of seven individuals using next-generation sequencing. We find that, on average, an iPSC line manifests two copy number variants (CNVs) not apparent in the fibroblasts from which the iPSC was derived. Using PCR and digital droplet PCR, we show that at least 50% of those CNVs are present as low-frequency somatic genomic variants in parental fibroblasts (that is, the fibroblasts from which each corresponding human iPSC line is derived), and are manifested in iPSC lines owing to their clonal origin. Hence, reprogramming does not necessarily lead to de novo CNVs in iPSCs, because most of the line-manifested CNVs reflect somatic mosaicism in the human skin. Moreover, our findings demonstrate that clonal expansion, and iPSC lines in particular, can be used as a discovery tool to reliably detect low-frequency CNVs in the tissue of origin. Overall, we estimate that approximately 30% of the fibroblast cells have somatic CNVs in their genomes, suggesting widespread somatic mosaicism in the human body. Our study paves the way to understanding the fundamental question of the extent to which cells of the human body normally acquire structural alterations in their DNA post-zygotically.

  15. [The influence of low-frequency pulsed electric and magnetic signals or their combination on the normal and modified fibroblasts (an experimental study)].

    Science.gov (United States)

    Ulitko, M V; Medvedeva, S Yu; Malakhov, V V

    2016-01-01

    The results of clinical studies give evidence of the beneficial preventive and therapeutic effects of the «Tiline-EM» physiotherapeutic device designed for the combined specific treatment of the skin regions onto which both discomfort and pain sensations are directly projected, reflectively active sites and zones, as well as trigger zones with the use of low-frequency pulsed electric current and magnetic field. The efficient application of the device requires the understanding of the general mechanisms underlying such action on the living systems including those operating at the cellular and subcellular levels. The objective of the present study was the investigation of the specific and complex effects produced by the low-frequency pulses of electric current and magnetic field generated in the physiotherapeutic device «Tiline-EM» on the viability, proliferative activity, and morphofunctional characteristics of normal skin fibroblasts and the transformed fibroblast line K-22. It has been demonstrated that the biological effects of the electric and magnetic signals vary depending on the type of the cell culture and the mode of impact. The transformed fibroblasts proved to be more sensitive to the specific and complex effects of electric and magnetic pulses than the normal skin fibroblasts. The combined action of the electric and magnetic signals was shown to have the greatest influence on both varieties of fibroblasts. It manifests itself in the form of enhanced viability, elevated proliferative and synthetic activity in the cultures of transformed fibroblasts and as the acceleration of cell differentiation in the cultures of normal fibroblasts. The effect of stimulation of dermal fibroblast differentiation in response to the combined treatment by the electric and magnetic signals is of interest from the standpoint of the physiotherapeutic use of the «Tiline-EM» device for the purpose of obtaining fibroblasts cultures to be employed in regenerative therapy and

  16. Remodeling of the fibroblast cytoskeletal architecture during the replication cycle of Ectromelia virus: A morphological in vitro study in a murine cell line.

    Science.gov (United States)

    Szulc-Dabrowska, Lidia; Gregorczyk, Karolina P; Struzik, Justyna; Boratynska-Jasinska, Anna; Szczepanowska, Joanna; Wyzewski, Zbigniew; Toka, Felix N; Gierynska, Malgorzata; Ostrowska, Agnieszka; Niemialtowski, Marek G

    2016-08-01

    Ectromelia virus (ECTV, the causative agent of mousepox), which represents the same genus as variola virus (VARV, the agent responsible for smallpox in humans), has served for years as a model virus for studying mechanisms of poxvirus-induced disease. Despite increasing knowledge on the interaction between ECTV and its natural host-the mouse-surprisingly, still little is known about the cell biology of ECTV infection. Because pathogen interaction with the cytoskeleton is still a growing area of research in the virus-host cell interplay, the aim of the present study was to evaluate the consequences of ECTV infection on the cytoskeleton in a murine fibroblast cell line. The viral effect on the cytoskeleton was reflected by changes in migration of the cells and rearrangement of the architecture of tubulin, vimentin, and actin filaments. The virus-induced cytoskeletal rearrangements observed in these studies contributed to the efficient cell-to-cell spread of infection, which is an important feature of ECTV virulence. Additionally, during later stages of infection L929 cells produced two main types of actin-based cellular protrusions: short (actin tails and "dendrites") and long (cytoplasmic corridors). Due to diversity of filopodial extensions induced by the virus, we suggest that ECTV represents a valuable new model for studying processes and pathways that regulate the formation of cytoskeleton-based cellular structures. © 2016 Wiley Periodicals, Inc.

  17. 脂多糖对人皮肤成纤维细胞胶原代谢的影响%Influence of lipopolysaccharide on collagen metabolism of normal skin fibroblasts of human

    Institute of Scientific and Technical Information of China (English)

    李凤玉; 王舒琦; 贾国洪; 万立; 杨红明

    2012-01-01

    Objective To observe the influence of lipopolysaccharide (LPS) on collagen metabolism of normal human skin fibroblasts and its biological role in the formation of hypertrophic scar.Methods Fibroblasts were isolated and cultured in vitro,and then exposed to different doses of LPS (0.005,0.01,0.05,0.1,0.5,1.0 μg/ml) from E.coli.055:B5 respectively.The expression of proccllagen type Ⅰ,Ⅲand collagenase mRNAs was tested by RT -PCR.Fibroblasts from hypertrophic scar tissue obtained from the same patients in the same culture passage were used as control.Results Compared with control group,the expression of procollagen type Ⅰ,Ⅲ mRNAs in normal skin fibroblasts increased (0.323 ± 0.041,0.303 ± 0.063,0.391 ± 0.071,0.344 ± 0.086,0.488 ± 0.059,0.401 ± 0.087,0.616 ± 0.107,0.434 ±0.084,0.823 ±0.092,0.542 ± 0.082),while the expression of collagenase mRNAs of normal skin fibroblasts depressed(0.598 ± 0.068,0.556 ± 0.049,0.441 ± 0.043,0.372 ± 0.083,0.260 ± 0.027 ).When LPS was set to the concentration of 0.005 μg/ml,it showed a concentration dependent manner.However,when the concentration of LPS was set to 0.5 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts began to decrease (0.451 ± 0.063,0.374 ± 0.072,0.360 ± 0.062).When the concentration of LPS was set to 1.0 μg/ml,the expression of procollagen type Ⅰ,Ⅲ mRNAs (0.162 ± 0.025,0.171 ± 0.061 )were inhibited and the expression of collagenase mRNAs began to increase (0.444 ±0.114).When the concentration of LPS was set to 0.1 μg/ml,the expression of procollagen type Ⅰ,Ⅲ and collagenase mRNAs of normal skin fibroblasts(0.823 ±0.092,0.542 ±0.082,0.260 ±0.027)was similar to that of hypertrophic scar tissue fibroblasts(0.829 ±0.049,0.569 ±0.038,0.277 ±0.059).Conclusions This result supported that LPS may be an important factor in collagen metabolism of normal skin fibroblasts and it plays an important role in hypertrophic scar formation.%目

  18. Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering*

    Science.gov (United States)

    Han, Chun-mao; Zhang, Li-ping; Sun, Jin-zhang; Shi, Hai-fei; Zhou, Jie; Gao, Chang-you

    2010-01-01

    To create a scaffold that is suitable for the construction of tissue-engineered skin, a novel asymmetric porous scaffold with different pore sizes on either side was prepared by combining a collagen-chitosan porous membrane with fibrin glue. Tissue-engineered skin was fabricated using this asymmetric scaffold, fibroblasts, and a human keratinocyte line (HaCaT). Epidermal cells could be seen growing easily and achieved confluence on the fibrin glue on the upper surface of the scaffold. Scanning electron microscopy showed typical shuttle-like fibroblasts adhering to the wall of the scaffold and fluorescence microscopy showed them growing in the dermal layer of the scaffold. The constructed composite skin substitute had a histological structure similar to that of normal skin tissue after three weeks of culture. The results of our study suggest that the asymmetric scaffold is a promising biologically functional material for skin tissue engineering, with prospects for clinical applications. PMID:20593518

  19. Application of collagen-chitosan/fibrin glue asymmetric scaffolds in skin tissue engineering.

    Science.gov (United States)

    Han, Chun-mao; Zhang, Li-ping; Sun, Jin-zhang; Shi, Hai-fei; Zhou, Jie; Gao, Chang-you

    2010-07-01

    To create a scaffold that is suitable for the construction of tissue-engineered skin, a novel asymmetric porous scaffold with different pore sizes on either side was prepared by combining a collagen-chitosan porous membrane with fibrin glue. Tissue-engineered skin was fabricated using this asymmetric scaffold, fibroblasts, and a human keratinocyte line (HaCaT). Epidermal cells could be seen growing easily and achieved confluence on the fibrin glue on the upper surface of the scaffold. Scanning electron microscopy showed typical shuttle-like fibroblasts adhering to the wall of the scaffold and fluorescence microscopy showed them growing in the dermal layer of the scaffold. The constructed composite skin substitute had a histological structure similar to that of normal skin tissue after three weeks of culture. The results of our study suggest that the asymmetric scaffold is a promising biologically functional material for skin tissue engineering, with prospects for clinical applications.

  20. 成纤维细胞激活基因的调控及改善皮肤老化的研究%Regulation of activated genes of fibroblasts and their improvement of skin aging

    Institute of Scientific and Technical Information of China (English)

    亓玉青; 李天航; 范丽云; 刘全忠

    2013-01-01

    目的 通过研究雌二醇、维生素E、维生素C对体外培养的人皮肤成纤维细胞基质金属蛋白酶-1(MMP-1) mRNA、透明质酸合成酶-2(HAS-2) mRNA转录水平的影响,探讨不同药物改善皮肤老化的可能机制.方法 将不同浓度的雌二醇、维生素E、维生素C分别加入体外培养的人皮肤成纤维细胞,逆转录-聚合酶链反应(RT-PCR)检测不同物质对成纤维细胞中HAS-2 mRNA、MMP-1 mRNA表达水平的影响.结果 雌二醇、维生素C对MMP 1 mRNA表达的影响:高剂量组、低剂量组与对照组的差异有统计学意义(P<0.05),低剂量组与高剂量组差异无统计学意义(P>0.05).维生素E、维生素C对HAS-2 mRNA表达的影响:高剂量组、低剂量组与对照组的差异有统计学意义(P<0.05),低剂量组与高剂量组差异无统计学意义(P>0.05).结论 雌二醇、维生素C可以下调MMP-1基因的转录水平,减少后者对皮肤胶原的降解;维生素E、维生素C可以上调HAS-2基因的转录水平,可能增加透明质酸的合成,从而改善皮肤老化.%Objective To investigate the possible mechanisms using different chemicals to improve the skin aging by the effect of estrogen,vitamin E,vitamin C on the transcriptional level of MMP-1 and HAS-2 mRNAs in cultured human skin fibroblast in vitro.Methods The different concentrations of estrogen,vitamin E,vitamin C were put into cultured human skin fibroblasts.After 24 hours,RT-PCR was used to detect the effects of the different chemicals on the transcriptional levels of the HAS-2 and MMP-1 mRNAs in fibroblasts.Results The effects of estrogen and vitamin C on the MMP-1 mRNA had significant difference between the high dose group and blank control group,and between the low dose group and blank control group (P<0.05) ; the difference between the low dose group and high dose group was not statistically significant (P>0.05).The effects of vitamin E and vitamin C on Has-2 mRNA also had significant

  1. Establishment and characteristics of a Yunnan pony ear marginal fibroblast cell line%云南矮马耳缘组织成纤维细胞系的建立及其生物学特性

    Institute of Scientific and Technical Information of China (English)

    周向梅; 马月辉; 关伟军; 赵德明

    2004-01-01

    A Yunnan pony ear marginal tissue fibroblast cell line (NYPEM 2/2) was successfully established using the explant of the ear marginal tissue and then trypsinization the cells from the outgrowth. Observations on cell morphology and dynamic growth, analysis of karyotype and isoenzymes of lactate dehydrogenase and malate dehydrogenase were carried out. The expression of recombinant green fluorescence protein in the cells were also undertaken. The results showed that the population doubling time (PDT) of the cells was 24 h; the frequency of cell chromosome number to be 2n = 64 was 92.9%; the banding patterns of the isozymes of the two enzymes had significant difference between the Yunnan pony ear marginal fibroblast cell line and the fibroblast cell lines of PEM 2/2, MSHEM 2/2 and BLCHE 2/2 derived from the Picdmont bovine ear, Mongolian ovine ear and Beijing local chicken embryo respectively. Tests for the contamination from bacteria, fungi or mycoplasma were negative; the transfection efficiency for the recombinant plasmid was 32.3%. This newly established cell line make the Yunnan pony breed, a national important genetic resource preserved at cell level, as well as will provide an effective experimental material for genetic studies on the Yunnan pony [ Acta Zoologica Sinica 50(5): 863-868, 2004].

  2. Evaluation of the effect of laser radiation on fibroblast proliferation in repair of skin wounds of rats with iron deficiency anemia

    Science.gov (United States)

    DeCastro, Isabele C. V.; Oliveira-Sampaio, Susana C. P.; Monteiro, Juliana S. de C.; Ferreira, Maria de Fátima L.; Cangussu, Maria T.; N. dos Santos, Jean; Pinheiro, Antonio Luiz B.

    2011-03-01

    The aim of this study was to assess the effect of low- level laser therapy (LLLT) on fibroblast proliferation on wound repair of rats with Iron deficiency anemia since there is no reports on literature about this subject. Iron deficiency anemia was induced on 36 newborn rats then an excisional wound was created on the dorsum of the animals which were divided into four groups: (I) - non-anemic, (II) - Anemic, (III) - non-anemic + LLLT, (IV) Anemic+ LLLT. The animals in each group were sacrificed at 7, 14 and 21 days. Laser irradiation was performed on each group (λ660nm,40Mw,CW) by contact mode with a dose of 2,5J/ cm2 in four points on the area of the wound and total of 10J/cm2 per session. Data were evaluated by analysis of variance (ANOVA) followed by Paired t-test. The results showed LLLT was able to stimulate fibroblastic proliferation in rats with iron deficiency anemia at the 21st day while at control group (III) no statistically significant differences was found.

  3. Inhibition of Mitochondrial Cytochrome c Release and Suppression of Caspases by Gamma-Tocotrienol Prevent Apoptosis and Delay Aging in Stress-Induced Premature Senescence of Skin Fibroblasts

    Directory of Open Access Journals (Sweden)

    Suzana Makpol

    2012-01-01

    Full Text Available In this study, we determined the molecular mechanism of γ-tocotrienol (GTT in preventing cellular aging by focusing on its anti-apoptotic effect in stress-induced premature senescence (SIPS model of human diploid fibroblasts (HDFs. Results obtained showed that SIPS exhibited senescent-phenotypic characteristic, increased expression of senescence-associated β-galactosidase (SA β-gal and promoted G0/G1 cell cycle arrest accompanied by shortening of telomere length with decreased telomerase activity. Both SIPS and senescent HDFs shared similar apoptotic changes such as increased Annexin V-FITC positive cells, increased cytochrome c release and increased activation of caspase-9 and caspase-3 (P<0.05. GTT treatment resulted in a significant reduction of Annexin V-FITC positive cells, inhibited cytochrome c release and decreased activation of caspase-9 and caspase-3 (P<0.05. Gene expression analysis showed that GTT treatment down regulated BAX mRNA, up-regulated BCL2A1 mRNA and decreased the ratio of Bax/Bcl-2 protein expression (P<0.05 in SIPS. These findings suggested that GTT inhibits apoptosis by modulating the upstream apoptosis cascade, causing the inhibition of cytochrome c release from the mitochondria with concomitant suppression of caspase-9 and caspase-3 activation. In conclusion, GTT delays cellular senescence of human diploid fibroblasts through the inhibition of intrinsic mitochondria-mediated pathway which involved the regulation of pro- and anti-apoptotic genes and proteins.

  4. Organotypic cocultures as skin equivalents: A complex and sophisticated in vitro system

    Directory of Open Access Journals (Sweden)

    Stark Hans-Jürgen

    2004-01-01

    Full Text Available To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and thus the precise analysis of growth regulatory mechanisms.

  5. Granulocyte macrophage colony stimulating factor (GM-CSF biological actions on human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    S Montagnani

    2009-12-01

    Full Text Available Fibroblasts are involved in all pathologies characterized by increased ExtraCellularMatrix synthesis, from wound healing to fibrosis. Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF is a cytokine isolated as an hemopoietic growth factor but recently indicated as a differentiative agent on endothelial cells. In this work we demonstrated the expression of the receptor for GM-CSF (GMCSFR on human normal skin fibroblasts from healthy subjects (NFPC and on a human normal fibroblast cell line (NHDF and we try to investigate the biological effects of this cytokine. Human normal fibroblasts were cultured with different doses of GM-CSF to study the effects of this factor on GMCSFR expression, on cell proliferation and adhesion structures. In addition we studied the production of some Extra-Cellular Matrix (ECM components such as Fibronectin, Tenascin and Collagen I. The growth rate of fibroblasts from healthy donors (NFPC is not augmented by GM-CSF stimulation in spite of increased expression of the GM-CSFR. On the contrary, the proliferation of normal human dermal fibroblasts (NHDF cell line seems more influenced by high concentration of GM-CSF in the culture medium. The adhesion structures and the ECM components appear variously influenced by GM-CSF treatment as compared to fibroblasts cultured in basal condition, but newly only NHDF cells are really induced to increase their synthesis activity. We suggest that the in vitro treatment with GM-CSF can shift human normal fibroblasts towards a more differentiated state, due or accompanied by an increased expression of GM-CSFR and that such “differentiation” is an important event induced by such cytokine.

  6. How to Choose the Best Skin Care Products

    Science.gov (United States)

    ... Lines and Forehead Furrows Hair Loss Hyperpigmentation Melasma Sagging Skin Scars Skin Growths Skin Lesions Spider Veins ... Lines and Forehead Furrows Hair Loss Hyperpigmentation Melasma Sagging Skin Scars Skin Growths Skin Lesions Spider Veins ...

  7. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    Science.gov (United States)

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  8. On-line dosimetry for photodynamic therapy: part I--incident irradiance versus space irradiance for human skin

    Science.gov (United States)

    Radasky, Christine J.; Crean, David H.; Doiron, Daniel R.

    1996-04-01

    One of the key components of photodynamic therapy dosimetry studies is the actual light dose delivered to the tissue at a targeted, therapeutic depth. The light dose delivered to the tissue is dependent upon the incident irradiance at the initial point of light application as well as the tissue optical properties. Since the parameters defining the light and therapeutic dose may vary between subjects and treatment sites as well as during treatment, it is advantageous to monitor the dosimetry during photodynamic therapy. The objective of this work is to develop methodologies and systems to enable on-line dosimetry. This initial study examines the relationship between incident irradiance and space irradiance for cutaneous areas in various humans. Space irradiance levels (mW/cm2) were measured on the skin surface of 30 subjects using isotropic fiber optic probes (0.8 mm) and were compared to incident irradiance levels. Space irradiance values were obtained from various anatomical locations on each subject during surface illumination (664 plus or minus 7 nm) using an incident irradiance of 100 mW/cm2. The results demonstrate ratios of space irradiance: incident irradiance from 1.49 to 2.35 for all cutaneous areas. Abnormal skin features, such as scars, birthmarks, moles, freckles, etc., on the arm demonstrated ratios ranging from 1.58 to 1.84. Variances observed within and between subjects demonstrate the need for accurate dosimetry monitoring during therapy. A larger study is planned to fully characterize space irradiance variations in normal skin as well as cutaneous lesions in additional subjects.

  9. 尾加压素Ⅱ对皮肤成纤维细胞表达Ⅰ型胶原蛋白的影响%Effects of urotensin Ⅱ on the expression of type Ⅰ collagen in human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    罗丽敏; 李军; 刘菡; 刘劲松; 董晓; 党书毅

    2014-01-01

    Objective To evaluate the effect of a vasoactive substance urotensin Ⅱ on the expression of type Ⅰ collagen and migration of human skin fibroblasts,and to explore the underlying mechanisms of signal transduction.Methods Fibroblasts were isolated from human foreskin tissues and subjected to primary culture.After a series of subculture,fibroblasts were classified into several groups to be treated with different concentrations (10-10 to 10-6 mol/L) of urotensin lⅡ for 24 hours,urotensin Ⅱ of 10-s mol/L for different durations (0,4,12,24 hours),or pretreated with PD98059 (a mitogen-activated protein kinase kinase inhibitor),nicardipine (a calcium channel blocker) and ciclosporin (a calcineurin inhibitor) of 10-5 mol/L respectively for 30 minutes followed by treatment with urotensin Ⅱ of 10-8 mol/L for 24 hours.The cells receiving no treatment served as the control.Subsequently,enzyme-linked immunosorbent assay was performed to determine the level of urotensin Ⅱ in the supernatant of fibroblasts,and Transwell assay to estimate the migration activity of fibroblasts.Statistical analysis was carried out by t test and analysis of variance.Results Urotensin Ⅱ promoted the expression of type Ⅰ collagen in a time-and concentrationdependent manner.The level of type Ⅰ collagen was increased by 21.2% (P > 0.05),52.2% (P < 0.05),84.4% (P <0.05),83.6% (P < 0.05) and 77.1% (P < 0.05) in the supernatant of fibroblasts treated with 10-10,10-9,10-8,10-7 and 10-6 mol/L of urotensin Ⅱ for 24 hours respectively,by 23.2% (P > 0.05),69.5% (P < 0.05) and 84.1% (P <0.05) in the supernatant of fibroblasts treated with urotensin Ⅱ of 10-8 mol/L for 4,12 and 24 hours respectively,compared with the untreated control fibroblasts.The migration activity was markedly enhanced in fibroblasts treated with urotensin Ⅱ of 10-8 mol/L for 24 hours compared with the control fibroblasts (P < 0.05).PD98059,nicardipine and cyclosporin A inhibited the

  10. Linkages between the life-history evolution of tropical and temperate birds and the resistance of cultured skin fibroblasts to oxidative and non-oxidative chemical injury.

    Science.gov (United States)

    Jimenez, Ana Gabriela; Harper, James M; Queenborough, Simon A; Williams, Joseph B

    2013-04-15

    A fundamental challenge facing physiological ecologists is to understand how variation in life history at the whole-organism level might be linked to cellular function. Thus, because tropical birds have higher annual survival and lower rates of metabolism, we hypothesized that cells from tropical species would have greater cellular resistance to chemical injury than cells from temperate species. We cultured dermal fibroblasts from 26 tropical and 26 temperate species of birds and examined cellular resistance to cadmium, H(2)O(2), paraquat, thapsigargin, tunicamycium, methane methylsulfonate (MMS) and UV light. Using ANCOVA, we found that the values for the dose that killed 50% of cells (LD(50)) from tropical birds were significantly higher for H(2)O(2) and MMS. When we tested for significance using a generalized least squares approach accounting for phylogenetic relationships among species to model LD(50), we found that cells from tropical birds had greater tolerance for Cd, H(2)O(2), paraquat, tunicamycin and MMS than cells from temperate birds. In contrast, tropical birds showed either lower or no difference in tolerance to thapsigargin and UV light in comparison with temperate birds. These findings are consistent with the idea that natural selection has uniquely fashioned cells of long-lived tropical bird species to be more resistant to forms of oxidative and non-oxidative stress than cells from shorter-lived temperate species.

  11. A Comparison between the Colony Formation of Adult Mouse Spermatogonial Stem Cells in Co cultures with Sertoli and STO (Mouse Embryonic Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Seyed Morteza Koruji

    2010-01-01

    Full Text Available Objective: The aim of this study was to compare the colony formation of spermatogonialstem cells (SSCs on sertoli and STO (Mouse embryonic fibroblast cell line feeder celllayers during a two-week period.Materials and Methods: Initially, sertoli cells and SSCs were isolated from adultmouse testes using a two-step enzymatic digestion and lectin immobilization. Characteristicsof the isolated cells were immunocytochemically confirmed by examiningfor the presence of Oct-4, CDH1, promyelocytic leukaemia zinc finger factor (PLZF,SSC C-kit, and the distribution of Sertoli cell vimentin. SSCs were then cultured abovethe Sertoli, STO and the control (without co-culture separately for two weeks. In allthree groups, the number and diameter of colonies were evaluated using an invert microscopeon the 3rd, 7th, 10th and 14th day. β1 and α6-integrin m-RNA expressions wereassessed using a reverse transcription polymerase chain reaction (RT-PCR and realtimePCR. Furthermore, Oct-4 m RNA expression was assessed using real time PCR.Statistical analysis was performed using ANOVA; and the paired two-sample t test andTukey’s test were used as post-hoc tests for the data analysis of the three sertoli, STOand control cocultures.Results: At the four specified time points, our results showed significant differences (p<0.05in colony numbers and diameters among the sertoli, STO and control groups. The numberand diameter of colonies increased more rapidly in the sertoli coculture than in the othertwo Our results at all four time points also showed significant differences (p<0.05 in themean colony numbers and diameters between the three groups, with the Sertoli coculturehaving the highest mean values for colony numbers and diameters. The RT-PCR results,after two-weeks of culturing, showed that β1-integrin was expressed in all three groups cocultures,but α6-integrin was not expressed. Additionally, based on real time PCR results,the three genes (β1-integrin, α6-integrin

  12. Effect of mitomycin on normal dermal fibroblast and HaCat cell: an in vitro study

    Institute of Scientific and Technical Information of China (English)

    Yao-wen WANG; Ji-hao REN; Kun XIA; Shu-hui WANG; Tuan-fang YIN; Ding-hua XIE; Li-hua LI

    2012-01-01

    Objective: To evaluate the effects of mitomycin on the growth of human dermal fibroblast and immortalized human keratinocyte line (HaCat cell),particularly the effect of mitomycin on intracellular messenger RNA (mRNA) synthesis of collagen and growth factors of fibroblast.Methods: The normal dermal fibroblast and HaCat cell were cultured in vitro.Cell cultures were exposed to 0.4 and 0.04 mg/ml of mitomycin solution,and serum-free culture medium was used as control.The cellular morphology change,growth characteristics,cell proliferation,and apoptosis were observed at different intervals.For the fibroblasts,the mRNA expression changes of transforming growth factor (TGF)-β1,basic fibroblast growth factor (bFGF),procollagen Ⅰ,and Ⅲ were detected by reverse transcription polymerase chain reaction (RT-PCR).Results: The cultured normal human skin fibroblast and HaCat cell grew exponentially.A 5-min exposure to mitomycin at either 0.4 or 0.04 mg/ml caused marked dose-dependent cell proliferation inhibition on both fibroblasts and HaCat cells.Cell morphology changed,cell density decreased,and the growth curves were without an exponential phase.The fibroblast proliferated on the 5th day after the 5-min exposure of mitomycin at 0.04 mg/ml.Meanwhile,5-min application of mitomycin at either 0.04 or 0.4 mg/ml induced fibroblast apoptosis but not necrosis.The apoptosis rate of the fibroblast increased with a higher concentration of mytomycin (p<0.05).A 5-min exposure to mitomycin at 0.4 mg/ml resulted in a marked decrease in the mRNA production of TGF-β1,procollagen Ⅰ and Ⅲ,and a marked increase in the mRNA production of bFGF.Conclusions: Mitomycin can inhibit fibroblast proliferation,induce fibroblast apoptosis,and regulate intracellular protein expression on mRNA levels.In additon,mitomycin can inhibit HaCat cell proliferation,so epithelial cell needs more protecting to avoid mitomycin's side effect when it is applied clinically.

  13. Effects of beta interferon on human fibroblasts at different population doubling levels. Proliferation, cell volume, thymidine uptake, and DNA synthesis

    OpenAIRE

    1984-01-01

    Cellular aging had no effect on the ability of beta interferon to increase cell volume and population doubling time in 76-109 cells, a line of human skin fibroblasts. However, DNA synthesis in cells at high population doubling levels (PDL 55-70) was inhibited after 72 h of beta interferon treatment (1,000 U/ml) while no inhibition of DNA synthesis was observed in cells at middle population doubling levels (PDL 30-40).

  14. On-line diffusion profile of a lipophilic model dye in different depths of a hair follicle in human scalp skin.

    Science.gov (United States)

    Grams, Ylva Y; Whitehead, Lynne; Lamers, Gerda; Sturmann, Nico; Bouwstra, Joke A

    2005-10-01

    In skin and hair research, drug targeting to the hair follicle is of great interest in the treatment of skin diseases. The aim of this study is to visualize on-line the diffusion processes of a model fluorophore into the hair follicle at different depths using fresh human scalp skin and confocal laser scanning microscopy. Up to a depth of 500 microm in the skin, a fast increase of fluorescence is observed in the gap followed by accumulation of the dye in the hair cuticle. Penetration was also observed via the stratum corneum and the epidermis. Little label reached depths greater than 2000 microm. Fat cells accumulated the label fastest, followed by the cuticular area and the outer root sheath of the hair follicle. Sweat glands revealed very low staining, whereas the bulb at a depth of 4000 microm was visualized only by autofluorescence. From this study, we conclude that on-line visualization is a promising technique to access diffusion processes in deep skin layers even on a cellular level. Furthermore, we conclude that the gap and the cuticle play an important role in the initial diffusion period with the label in the cuticle originating from the gap.

  15. Development of an Ussuri catfish Pseudobagrus ussuriensis skin cell line displaying differential cytopathic effects to three aquatic animal viruses.

    Science.gov (United States)

    Ou, Tong; Lei, Xiao-Ying; He, Li-Bo; Zhou, Feng-Jian; Zhang, Qi-Ya

    2014-08-30

    An Ussuri catfish Pseudobagrus ussuriensis skin (UCS) cell line was developed and subcultured for more than 60 passages. UCS cells consisted of mostly epithelial-like cells and multiplied well in TC199 medium supplemented with 10% fetal bovine serum at 25°C. Chromosome analysis revealed that most UCS cells had a normal diploid karyotype with 2n=52. UCS cells showed differential cytopathic effects (CPEs) after inoculation of spring viremia of carp virus (SVCV, a negative-strand RNA virus), grass carp reovirus (GCRV, a multi-segmented double-stranded RNA virus) and Rana grylio virus (RGV, a large double-stranded DNA virus), and were indicative of high sensitivities to these three aquatic animal viruses by a virus titration study. The CPE caused by SVCV appeared as rounded and granular cells, grape-like clusters and small lytic plaques. Characteristic CPE containing plaque-like syncytia was induced by GCRV. RGV-infected cells produced typical CPE characterized by cells shrinkage and aggregation, formation of clear plaques and cell sheet detachment. Furthermore, significant fluorescent signals were observed after UCS cells were transfected with green fluorescent protein reporter plasmids, and the development of CPE induced by a recombinant RGV, ΔTK-RGV, in UCS cells was illustrated using a combination of light and fluorescence microscopy. The data from this study suggested that UCS cell line can potentially serve as a useful tool for the comparison study of different aquatic animal viruses and the isolation of some newly emerging viruses in Ussuri catfish farming.

  16. Application of Allogeneic Fibroblast Cells in Cellular Therapy of Recessive Dystrophic Epidermolysis Bullosa

    Directory of Open Access Journals (Sweden)

    Zare

    2015-09-01

    Full Text Available Context Connective tissue cells include fibroblasts, chondrocytes, adipocyte, and osteocytes. These cells are specialized for the secretion of collagenous extracellular matrix and are responsible for the architectural framework of the human body. Evidence Acquisition Connective tissue cells play a central role in supporting as well as repairing tissues and organs. Fibroblast cell therapy could be used for the treatment of burn wounds, scars, diabetic foot ulcers, acne scars and skin aging. This review focused on biology of fibroblasts and their role in cell therapy of recessive dystrophic epidermolysis bullosa (RDEB. Results Fibroblasts are known to play a pivotal role in skin structure and integrity, and dermal fibroblasts are believed to promote skin regeneration and rejuvenation via collagen production. Conclusions Fibroblasts can be used in transplantations to ameliorate an immune system response, in order to reduce antigen production. Human fibroblasts suppress ongoing mixed lymphocyte reactions (MLRs between lymphocyte cells from two individuals, and supernatant materials from fibroblast cultures suppress MLRs.

  17. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    Science.gov (United States)

    Sklirou, Aimilia D.; Ralli, Marianna; Dominguez, Maria; Papassideri, Issidora; Skaltsounis, Alexios-Leandros; Trougakos, Ioannis P.

    2015-01-01

    Despite the fact that several natural products (e.g. crude extracts or purified compounds) have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s) of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network) remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11) of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP) originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent. PMID:25974626

  18. Hexapeptide-11 is a novel modulator of the proteostasis network in human diploid fibroblasts

    Directory of Open Access Journals (Sweden)

    Aimilia D. Sklirou

    2015-08-01

    Full Text Available Despite the fact that several natural products (e.g. crude extracts or purified compounds have been found to activate cell antioxidant responses and/or delay cellular senescence the effect(s of small peptides on cell viability and/or modulation of protective mechanisms (e.g. the proteostasis network remain largely elusive. We have thus studied a hexapeptide (Hexapeptide-11 of structure Phe–Val–Ala–Pro–Phe–Pro (FVAPFP originally isolated from yeast extracts and later synthesized by solid state synthesis to high purity. We show herein that Hexapeptide-11 exhibits no significant toxicity in normal human diploid lung or skin fibroblasts. Exposure of fibroblasts to Hexapeptide-11 promoted dose and time-dependent activation of proteasome, autophagy, chaperones and antioxidant responses related genes. Moreover, it promoted increased nuclear accumulation of Nrf2; higher expression levels of proteasomal protein subunits and increased proteasome peptidase activities. In line with these findings we noted that Hexapeptide-11 conferred significant protection in fibroblasts against oxidative-stress-mediated premature cellular senescence, while at in vivo skin deformation assays in human subjects it improved skin elasticity. Finally, Hexapeptide-11 was found to induce the activity of extracellular MMPs and it also suppressed cell migration. Our presented findings indicate that Hexapeptide-11 is a promising anti-ageing agent.

  19. Growth of fibroblasts and endothelial cells on wettability gradient surfaces

    NARCIS (Netherlands)

    Ruardy, TG; Moorlag, HE; Schakenraad, JM; VanderMei, HC; Busscher, HJ

    1997-01-01

    The growth, spreading, and shape of human skin fibroblasts (PK 84) and human umbilical cord endothelial cells on dichlorodimethylsilane (DDS) and dimethyloctadecylchlorosilane (DOGS) gradient surfaces were investigated in the presence of serum proteins. Gradient surfaces were prepared on glass using

  20. Parental somatic and germ-line mosaicism for a FBN2 mutation and analysis of FBN2 transcript levels in dermal fibroblasts.

    Science.gov (United States)

    Putnam, E A; Park, E S; Aalfs, C M; Hennekam, R C; Milewicz, D M

    1997-01-01

    Congenital contractural arachnodactyly (CCA) is an autosomal dominant disorder that is phenotypically related to the Marfan syndrome. CCA has recently been shown to result from mutations in the FBN2 gene, which encodes an elastin-associated microfibrillar protein called fibrillin-2. Two siblings are reported here with classic manifestations of CCA with unaffected parents. Analysis of the FBN2 cDNA from dermal fibroblasts from one of the affected siblings revealed a heterozygous exon splicing error deleting nt 3722-3844 of the FBN2 mRNA. This cDNA deletion resulted in selective removal of one of the 43 calcium-binding EGF-like domains of the fibrillin-2 protein. Analysis of the FBN2 gene in the affected siblings' DNA indicated that the splicing error resulted from an A-to-G transition 15 nt upstream from the 3' splice site of the intron. The genomic mutation resulting in the splicing error alters a putative branch point sequence important for lariat formation, an intermediate structure of normal splicing. The mutation was detectable in DNA from the father's hair bulbs and buccal cells but not his white blood cell DNA, indicating that the father was a somatic mosaic. Analysis of transcript levels by use of dermal fibroblasts from the proband demonstrated that the FBN2 allele containing the exon deletion was expressed at a higher level than the allele inherited from the mother. These results indicate that FBN2 exon splicing errors are a cause of CCA, furthering the understanding of the molecular basis of this disorder. In addition, the demonstration of gonadal mosaicism in the FBN2 gene is important for accurate genetic counseling of families with sporadic cases of CCA. Finally, the preferential expression of the mutated FBN2 allele in dermal fibroblasts may have implications for understanding the pathogenesis and rarity of CCA. Images Figure 1 Figure 3 Figure 4 PMID:9106527

  1. Manganese superoxide dismutase: effect of the ala16val polymorphism on protein, activity, and mRNA levels in human breast cancer cell lines and stably transfected mouse embryonic fibroblasts.

    Science.gov (United States)

    McAtee, Britt L; Yager, James D

    2010-02-01

    The manganese superoxide dismutase (MnSOD) ala16val polymorphism has been associated with various diseases including breast cancer. In the present study, we investigated levels of MnSOD protein, enzymatic activity, and mRNA with respect to MnSOD genotype in several human breast carcinoma cell lines and in mouse embryonic fibroblasts (MEF), developed from the MnSOD knockout mouse, stably expressing human MnSOD-ala and MnSOD-val. In human breast cell lines, the MnSOD-ala allele was associated with increased levels of MnSOD protein and MnSOD protein per unit mRNA. In the MEF transformants, MnSOD activity correlated fairly well with MnSOD protein levels. MnSOD mRNA expression was significantly lower in MnSOD-ala versus MnSOD-val lines. MnSOD protein and activity levels were not related to MnSOD genotype in the transformed MEF, although, as observed in the human breast cell lines, the MEF human MnSOD-ala lines produced significantly more human MnSOD protein per unit mRNA than the human MnSOD-val lines. This suggests that there is more efficient production of MnSOD-ala protein compared to MnSOD-val protein. Examination of several indicators of reactive oxygen species levels, including superoxide and hydrogen peroxide, in wild-type MEF and in MEF expressing similar elevated amounts of MnSOD-ala or val activity did not show differences related to the levels of MnSOD protein expression. In conclusion, in both human breast carcinoma cell lines and MEF cell lines stably transfected with human MnSOD, the MnSOD-ala allele was associated with increased production of MnSOD protein per unit mRNA indicating a possible imbalance in MnSOD protein production from the MnSOD-val mRNA.

  2. Establishment and Biological Characteristic Analysis of Fetal Fibroblast Cell Line for Jinhua Pig%金华猪胎儿成纤维细胞系的建立与生物学特性分析

    Institute of Scientific and Technical Information of China (English)

    郝柱; 王颖; 彭静; 沈一飞; 郭晓令; 徐宁迎

    2012-01-01

    建立并保存家畜的成纤维细胞在保护畜禽种质资源研究中发挥着重要作用.本研究以40日龄金华猪胚胎为实验材料,采用组织贴壁法建立了金华猪胎儿成纤维细胞系,并对所建细胞系进行生物学特性研究.结果表明,原代细胞生长迅速,贴壁后2~3d可长满培养瓶,获得的成纤维细胞生长态势良好;细胞生长曲线为典型的S形,细胞群体倍增时间(population doubling time,PDT)约为36h;细胞冻存前、复苏后的活率分别为95.2%和92.8%;细胞中期染色体二倍体(2n=38)占主体约为91%,达到了建立成纤维细胞系的要求;苹果酸脱氢同工酶(malic dehydrogenase,MDH)和乳酸脱氢同工酶(lactic dehydrogenase,LDH)电泳结果表明,本细胞系没有被其他细胞污染,细胞纯度较高;细菌、真菌和支原体三类微生物检测结果均为阴性,细胞没有受到微生物的污染;15代细胞的凋亡率为2.0%,细胞没有大规模的凋亡现象发生;当阳离子脂质体(Lipofectamine 2000)剂量为0.3 μL、荧光蛋白报告质粒(pEGFP-N3)为0.5 μg时转染成纤维细胞的效率最高,可达32.4%.细胞系各项指标均达到美国典型培养中心(American Type Culture Collection,ATCC)标准.金华猪胎儿成纤维细胞系的成功建立使金华猪种质资源在细胞水平得到保存,并可为胚胎克隆以及转基因等研究提供必要的实验材料;也可以为以后其他种质资源在细胞水平上的保存提供理论与技术支持.%Establishment and preservation of livestock fibroblasts play a important role in protecting of animal genetic resources. The fetal fibroblast cell line of Jinhua pig was successfully established from 40 d's embryos by direct explant technique, and the cell morphology, growth dynamic, vitality, chromosome, isoenzymes, microbial inspection, cell apoptosis, as well as the transfection of fluorescent protein report plasmid (pEGFP-N3) had been studied in the line. The

  3. Governance of Cutaneous Photocarcinogenesis by Chronic UVA-Exposed Dermal Fibroblasts

    Science.gov (United States)

    2014-09-01

    fibroblasts and determine if bystander oxidative stress is induced by UVA irradiation and abrogated by antioxidants . In initial studies, we selected...primary human skin cells in order to avoid untoward effects of immortalization or transformation. Initial experiments determined direct effects...of UVA on normal human melanocytes (NHMs), fibroblasts (NHFs) and keratinocytes (NHKs). Figure 1A shows dose- dependent toxicity for three skin

  4. EFFECT OF TASPINE ON WOUND HEALING AND FIBROBLAST PROLIFERATION

    Institute of Scientific and Technical Information of China (English)

    Dong Yalin; He Langchong; Chen Fang

    2005-01-01

    Objective To study the effect and mechanism of taspine on wound healing and fibroblast proliferation. Methods The effect of taspine on skin wound was observed in vivo. The different concentration of taspine hydrochloride was added to L929 fibroblast cultivated in vitro, and lactate dehydrogenase was detected and MTT method was applied to observe effect of taspine on fibroblast proliferation. Results The local application of taspine 3 mg/Ml and 1.5 mg/mL accelerated the healing of skin wounded. In vitro, 0.01~0.5 μg/mL of taspine hydrochloride showed no effect on the change of lactate dehydrogenase activity and fibroblast proliferation. Conclusion Taspine is a kind of active alkaloid from leontice robustum which can enhance wound healing, its mechanism on wound healing is not by means of accelerating the proliferation of fibroblast, other mechanisms are necessary for being further studied.

  5. PAMPs and DAMPs stimulate the expression of pro-inflammatory cytokines in vitro in a fibroblast cell-line from rainbow trout (Oncorhynchus mykiss)

    DEFF Research Database (Denmark)

    Ingerslev, Hans-Christian; Ossum, C.G.; Nielsen, Michael Engelbrecht

    activates downstream signalling pathways, which subsequently leads to expression of pro-inflammatory cytokines and chemokines. DAMPs released from necrotic cells may also bind to and activate similar downstream signalling events. In telosts was found that mechanical damage of the muscle tissue using sterile...... needles induced a very rapid expression of the pro-inflammatory cytokines IL-1β, IL-8 and IL-10 as measured by real-time PCR. The results imply that cells located in the muscular tissue in addition to recruited cells are involved in the observed increased cytokine / chemokine expression. It is believed...... in this evolutionary lineage of the bony fishes. The expression of TLR-3 and -9 receptors were significantly up-regulated following physical damage of muscle tissue as well as in stimulated fibroblasts, where LPS induced both TLR-3 and -9, supernatant from sonicated cells only TLR-9 while debris caused no induction...

  6. Dexamethasone prevents granulocyte-macrophage colony-stimulating factor-induced nuclear factor-κB activation, inducible nitric oxide synthase expression and nitric oxide production in a skin dendritic cell line

    Directory of Open Access Journals (Sweden)

    Ana Luísa Vital

    2003-01-01

    Full Text Available Aims: Nitric oxide (NO has been increasingly implicated in inflammatory skin diseases, namely in allergic contact dermatitis. In this work, we investigated the effect of dexamethasone on NO production induced by the epidermal cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF in a mouse fetal skin dendritic cell line.

  7. Inhibitory Effects of Baicalin on Formation of the Photoproduct in Human Skin Fibroblasts Induced by Ultraviolet B Light%黄芩苷对UVB诱导人皮肤成纤维细胞光产物生成的干预作用

    Institute of Scientific and Technical Information of China (English)

    吴迪; 周炳荣; 骆丹

    2011-01-01

    Objective To observe the inhibitory effect of Baicalin on formation of the photoproduct in human skin fibroblasts induced by ultraviolet B (UVB) light. Methods Human skin fibroblasts were incubated with various concentrations of Baicalin (0-250μg/mL) for 24 hours. Immunohistochemistry and immune dot blot test (IDBT) was used to detect cyclobutane pyrimidine dimmers (CPDs) in DNA of human skin fibroblasts 30 minutes after 30mJ/cm2 UVB radiation and Baicalin intervention. The UV absorbance ability of Baicalin was detected by UV spectrophotometer. Results Baicalin could reduced CPDs formation 30 minutes after UVB radiation. Baicalin had a strong ability to absorb ultraviolet energy within UVA, UVB and UVC band. Conclusion Baicalin can inhibit the the formation of the photoproducts induced by UVB light in human skin fibroblasts. Baicalin is an effective UV protectant.%目的 观察中波紫外线(UVB)诱导人皮肤成纤维细胞光产物的形成情况,以及黄芩苷的干预作用.方法将各种不同浓度的黄芩苷(0~250μg/mL)与人皮肤成纤维细胞共同孵育24h后,采用免疫组织化学法及免疫斑点印迹技术检测30mJ/cm2UVB辐射及黄芩苷干预下,辐射后30min人皮肤成纤维细胞DNA内环丁烷嘧啶二聚体( CPDs)的产生情况.并采用紫外分光光度计法检测黄芩苷在紫外波段的吸光度.结果黄芩苷能在UVB辐射后30min内减轻光产物的形成,其在UVA、UVB和UVC波段内有较强的吸收紫外线能量的能力.结论黄芩苷对UVB诱导人皮肤成纤维细胞光产物的形成有一定抑制作用.黄芩苷是一种有效的紫外线防护剂.

  8. Positive correlation between the efficiency of induced pluripotent stem cells and the development rate of nuclear transfer embryos when the same porcine embryonic fibroblast lines are used as donor cells.

    Science.gov (United States)

    Xie, Bingteng; Wang, Jianyu; Liu, Shichao; Wang, Jiaqiang; Xue, Binghua; Li, Jingyu; Wei, Renyue; Zhao, Yanhua; Liu, Zhonghua

    2014-06-01

    Induced pluripotent stem cells (iPSCs) and nuclear transfer (NT) are two of the primary routes to reprogram differentiated cells back to the pluripotent state. However, it is still unknown whether there is any correlation between the reprogramming efficiency of iPSCs and NT if the same donor cells are employed. In this study, six porcine embryonic fibroblast (PEF) lines from Landrace (L1, L6, L9) or Congjiang local pigs (C4, C5, C6) were used for iPSC induction and NT. Furthermore, the resultant iPSCs from four PEF lines (L1, L6, C4, and C5) were used for NT (iPSC-NT), and the expression of exogenous genes was detected in iPSC-NT embryos by real-time PCR. The results showed that the efficiency of iPSC lines established from different PEF lines were significantly different. When the same PEF lines were used as donor cells for NT, the blastocysts rates were also different among different PEF lines and positively related with iPSCs induction efficiency. When the iPSCs were used as donor cells for NT, compared with the source PEFs, the blastocysts rates were significantly decreased. Real-time PCR results indicated that exogenous genes (Oct4, c-Myc) continued to be expressed in iPSC-NT embryos. In summary, our results demonstrate that there was a positive correlation between iPSCs and NT reprogramming efficiency, although the mechanism of these two routes is different. This may provide a new method to select the appropriate donor cells for inducing iPSCs.

  9. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    Directory of Open Access Journals (Sweden)

    Agnieszka Śmieszek

    2015-01-01

    Full Text Available Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs and Balb/3T3 fibroblast at concentration of 1 mM, 5 mM, and 10 mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2 signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10 mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5 mM and increased after 10 mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure.

  10. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF) Cell Lines.

    Science.gov (United States)

    Santhanam, Ramesh Kumar; Ahmad, Syahida; Abas, Faridah; Safinar Ismail, Intan; Rukayadi, Yaya; Tayyab Akhtar, Muhammad; Shaari, Khozirah

    2016-05-24

    Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  11. Bioactive Constituents of Zanthoxylum rhetsa Bark and Its Cytotoxic Potential against B16-F10 Melanoma Cancer and Normal Human Dermal Fibroblast (HDF Cell Lines

    Directory of Open Access Journals (Sweden)

    Ramesh Kumar Santhanam

    2016-05-01

    Full Text Available Zanthoxylum rhetsa is an aromatic tree, known vernacularly as “Indian Prickly Ash”. It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin, a berberine alkaloid (columbamine and a triterpenoid (lupeol from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF and mouse melanoma (B16-F10 cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells.

  12. Effects of Essential Oils and Polyunsaturated Fatty Acids on Canine Skin Equivalents: Skin Lipid Assessment and Morphological Evaluation

    OpenAIRE

    Cerrato, S.; Ramió-Lluch, L.; Fondevila, D.; Rodes, D.; P. Brazis; Puigdemont, A.

    2013-01-01

    A canine skin equivalent model has been validated for the assessment of a topical formulation effects. Skin equivalents were developed from freshly isolated cutaneous canine fibroblasts and keratinocytes, after enzymatic digestion of skin samples (n = 8) from different breeds. Fibroblasts were embedded into a collagen type I matrix, and keratinocytes were seeded onto its surface at air-liquid interface. Skin equivalents were supplemented with essential oils and polyunsaturated fatty acid form...

  13. Impact of the neutron and nuclear matter equations of state on neutron skin and neutron drip lines in chiral effective field theory

    CERN Document Server

    Sammarruca, Francesca

    2016-01-01

    We present predictions of the binding energy per nucleon and the neutron skin thickness in highly neutron-rich isotopes of Oxygen, Magnesium, and Aluminum. The calculations are carried out at and below the neutron drip line. The nuclear properties are obtained via an energy functional whose input is the equation of state of isospin-asymmetric in?finite matter. The latter is based on a microscopic derivation applying chiral few-nucleon forces. We highlight the impact of the equation of state at diff?erent orders of chiral effective fi?eld theory and discuss the role of three-neutron forces.

  14. Effects of pro-inflammatory cytokines on expression of kynurenine pathway enzymes in human dermal fibroblasts

    Directory of Open Access Journals (Sweden)

    Kegel Magdalena

    2011-10-01

    Full Text Available Abstract Background The kynurenine pathway (KP is the main route of tryptophan degradation in the human body and generates several neuroactive and immunomodulatory metabolites. Altered levels of KP-metabolites have been observed in neuropsychiatric and neurodegenerative disorders as well as in patients with affective disorders. The purpose of the present study was to investigate if skin derived human fibroblasts are useful for studies of expression of enzymes in the KP. Methods Fibroblast cultures were established from cutaneous biopsies taken from the arm of consenting volunteers. Such cultures were subsequently treated with interferon (IFN-γ 200 U/ml and/or tumor necrosis factor (TNF-α, 100 U/ml for 48 hours in serum-free medium. Levels of transcripts encoding different enzymes were determined by real-time PCR and levels of kynurenic acid (KYNA were determined by HPLC. Results At base-line all cultures harbored detectable levels of transcripts encoding KP enzymes, albeit with considerable variation across individuals. Following cytokine treatment, considerable changes in many of the transcripts investigated were observed. For example, increases in the abundance of transcripts encoding indoleamine 2,3-dioxygenase, kynureninase or 3-hydroxyanthranilic acid oxygenase and decreases in the levels of transcripts encoding tryptophan 2,3-dioxygenase, kynurenine aminotransferases or quinolinic acid phosphoribosyltransferase were observed following IFN-γ and TNF-α treatment. Finally, the fibroblast cultures released detectable levels of KYNA in the cell culture medium at base-line conditions, which were increased after IFN-γ, but not TNF-α, treatments. Conclusions All of the investigated genes encoding KP enzymes were expressed in human fibroblasts. Expression of many of these appeared to be regulated in response to cytokine treatment as previously reported for other cell types. Fibroblast cultures, thus, appear to be useful for studies of disease

  15. In vitro development competence of bovine nuclear transfer embryos derived from Nanog-overexpressing fibroblast cells

    Directory of Open Access Journals (Sweden)

    Xi-bang Zheng, Yan Yun, Yong-ce Hu, Yong Li, Hua-yan Wang, Xiao-ling Ma, Jin-qiang Sui, An-min Lei and Zhong-ying Dou

    2014-04-01

    Full Text Available The purpose of this study was to establish Nanog-expressing cell lines that can be used as donor cells to construct transgenic cloned embryos, and to investigate their in vitro development competence. By reverse transcription-polymerase chain reaction (RT-PCR, the cDNA of Nanog gene was cloned from fetal bovine primordial genital ridge tissues. The gene was inserted into PMD18-T vector using recombination techniques and then subcloned into vector pEGFP-C1. After confirmation by restrictive endonuclease digestion and sequencing, the recombinant plasmid pEGFP-Nanog was transfected into skin fibroblast cells. A stable transfected cell line was successfully established after two months of selection with neomycine (G418. Fluorescence microscopy, RT-PCR, and Western Blotting assays indicated that Nanog mRNA and EGFP-Nanog fusion protein were expressed in these cells. The EGFP-Nanog expressing fibroblast cells and the intact fibroblast cells (BEF422 were respectively used to construct cloned embryos. The results showed that the cleavage rate of recombinant embryos in BEF422 cells was significantly (P<0.05 higher than in EGFP-Nanog expressing cells (82.14 vs 40.38 %, but the blastocyst development rate in the latter was slightly higher than in the former (17.30 vs 14.29% (P<0.05, indicating that Nanog-overexpressed fibroblasts may be a better candidate of donor cells. To our knowledge, this is the first time that Nanog gene has been introduced into fibroblast cells to produce cloned embryos in bovine.

  16. Generation of GFAP::GFP astrocyte reporter lines from human adult fibroblast-derived iPS cells using zinc-finger nuclease technology.

    Science.gov (United States)

    Zhang, Ping-Wu; Haidet-Phillips, Amanda M; Pham, Jacqueline T; Lee, Youngjin; Huo, Yuqing; Tienari, Pentti J; Maragakis, Nicholas J; Sattler, Rita; Rothstein, Jeffrey D

    2016-01-01

    Astrocytes are instrumental to major brain functions, including metabolic support, extracellular ion regulation, the shaping of excitatory signaling events and maintenance of synaptic glutamate homeostasis. Astrocyte dysfunction contributes to numerous developmental, psychiatric and neurodegenerative disorders. The generation of adult human fibroblast-derived induced pluripotent stem cells (iPSCs) has provided novel opportunities to study mechanisms of astrocyte dysfunction in human-derived cells. To overcome the difficulties of cell type heterogeneity during the differentiation process from iPSCs to astroglial cells (iPS astrocytes), we generated homogenous populations of iPS astrocytes using zinc-finger nuclease (ZFN) technology. Enhanced green fluorescent protein (eGFP) driven by the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter was inserted into the safe harbor adeno-associated virus integration site 1 (AAVS1) locus in disease and control-derived iPSCs. Astrocyte populations were enriched using Fluorescence Activated Cell Sorting (FACS) and after enrichment more than 99% of iPS astrocytes expressed mature astrocyte markers including GFAP, S100β, NFIA and ALDH1L1. In addition, mature pure GFP-iPS astrocytes exhibited a well-described functional astrocytic activity in vitro characterized by neuron-dependent regulation of glutamate transporters to regulate extracellular glutamate concentrations. Engraftment of GFP-iPS astrocytes into rat spinal cord grey matter confirmed in vivo cell survival and continued astrocytic maturation. In conclusion, the generation of GFAP::GFP-iPS astrocytes provides a powerful in vitro and in vivo tool for studying astrocyte biology and astrocyte-driven disease pathogenesis and therapy.

  17. 撒坝猪成纤维细胞系的建立及其生物学特性分析%Establishment and the Biological Characteristics of Fibroblast Cell Line in Saba Pig

    Institute of Scientific and Technical Information of China (English)

    吕春荣; 权国波; 吴国权; 洪琼花

    2016-01-01

    In this study, the fibroblasts cell line derived from ear marginal tissue of saba pig was successfully established using the primary explants technique and cryopreservation technology. The morphology, cell survival rate and karyotypes of the cultured cells after different passages were analyzed, The tissue cells of the ear were observed by transmission electron microscopy.The results showed that the population doubling time ( PDT) of the cells was 48 h; The growth curve of the cultured cells appeared“S” shape; The frequency of cell chromosome number to be 2 n =38 was 92.56 %; Tests for the contamination from bacteria, fungi or mycoplasma were negative; Transmission electron microscopy showed that fibroblast cells were long shuttle type.%实验采用撒坝猪耳缘组织块贴壁培养法首次对撒坝猪成纤维细胞进行体外培养,通过原代培养、传代培养、冷冻保存建立了撒坝猪耳缘成纤维细胞系;并对其细胞形态、细胞活率、细胞核型等生物学特性进行分析;还利用透射电镜观察了耳缘组织细胞。结果表明:细胞倍增时间为48 h,生长曲线为“S”型;染色体中正常二倍体染色体(2n=38)所占比例为92.56%;细菌、真菌、病毒和支原体等微生物污染检测均显示为阴性。透射电镜观察显示,成纤维细胞呈长梭型。

  18. Multiple Directional Differentiation Difference of Neonatal Rat Fibroblasts from Six Organs

    Directory of Open Access Journals (Sweden)

    Yuqiao Chang

    2016-06-01

    Full Text Available Background/Aims: Fibroblasts are abundantly distributed throughout connective tissues in the body and are very important in maintaining the structural and functional integrity. Recent reports have proved that fibroblasts and mesenchymal stem cells share much more in common than previously recognized. The aim of this study was to investigate comparative studies in fibroblasts on the differences in the expression of molecular markers and differentiation capacity from different organs. Methods: Combined trypsin/collagenase enzymes digestion method was used to isolate and culture the fibroblasts derived from heart, liver, spleen, lung, kidney and skin. Cell activity was determined by methyl thiazolyl tetrazolium (MTT assay. Common molecular markers for fibroblasts such as vimentin, DDR2 and FSP1, stem cell markers nanog, c-kit and sca-1 were detected by RT-PCR, immunofluorescence and western blotting. The osteogenic, adipogenic and cardiogenic differentiations of fibroblasts were performed by inductive culture in special mediums, and analyzed by Alizarin red, Oil red O and immunofluorescence staining of cTnT respectively. Results: The proliferation rate of fibroblasts in lung was faster than in other five organs. Common molecular markers for fibroblasts were expressed differently in different organs. DDR2 was strongly expressed in fibroblasts in the heart, partly expressed in the heart, skin, liver and spleen. Interestingly, no expression of DDR2 was detected in liver and kidney. However, vimentin and FSP1 were consistently expressed in fibroblasts from skin, liver, kidney, spleen and lung. nanog expression in fibroblasts from lung was less than that from heart, skin, liver and spleen (P . c-kit expression in fibroblasts from heart, skin and kidney was higher than that from spleen (P , while the c-kit positive fibroblasts from liver was obviously higher than that from spleen (P . But sca-1 expression in fibroblasts from lung was the lowest among six

  19. UVB照射诱导皮肤成纤维细胞早期衰老的研究%Ultroviolet B exposure triggers premature senescence in human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    陈文琦; 许惠娟; 毕志刚

    2010-01-01

    目的 探讨UVB诱导的细胞衰老与肿瘤发生的关系.方法 噻唑蓝(MTT)法检测UVB辐射后的细胞增殖情况,筛选诱导衰老适宜的亚毒性剂量和照射次数.染色法检测衰老相关的β-半乳糖苷酶(SA β-gal)活性.RT-PCR检测衰老相关基因纤维结合素(FN)、骨结合素(ON)和平滑肌22(SM22)的表达.结果 以10 mJ/cm~2的亚毒性剂量连续5次照射人成纤维细胞后,衰老的生物学特征得以明显表现:①MTT法检测显示细胞增生能力的减弱.②照射组有SA β-gal活性的阳性细胞明显增加,照射组和对照组的阳性率分别为82.0%和33.7%(P<0.01).③3种衰老相关基因FN、ON和SM22的表达亦明显增强,分别约为对照组细胞的2.7、2.0、2.3倍(P<0.05).结论 反复亚毒性剂量UVB照射人成纤维细胞,初步建立一种UVB诱导的应激诱导的早期衰老(SIPS)模型.%Objective To develop a model of ultraviolet B (UVB)-induced premature senescence in human skin fibroblasts (HSF) so as to assess the relationship between stress-induced premature senescence and tumorigenesis. Methods The irradiation dose and frequency were optimized for the induction of prema-ture senescence. HSF were irradiated with UVB of 10 mJ/cm~2 once daily for 5 days, and unirradiated HSFs served as the control. After the last irradiation, cell proliferation was determined by MTT assay on day 3, 4, 5,6 and 7, SA β-Gal staining was performed to evaluate the senescence state of cells on day 3, and RT-PCR to detect the expressions of three senescene-associated genes, including fibronectin (FN), osteonectin (ON) and smooth muscle 22 (SM22) on day 3. Results After five exposures to UVB, HSF showed biological characteris-tics of senescence. As assessed by MTT assay, there was a loss of replicative potential in irradiated cells. The proportion of SA-β-gal-positive cells was 82.0% in UVB-stressed HSFs and 33.7% in the control cells (P < 0.01). The mRNA levels of FN, ON and SM22 were upregulated

  20. 人皮肤成纤维细胞来源诱导性多潜能干细胞的建立和鉴定%Generation of human induced pluripotent stem cells from human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    蒋知新; 张清华; 艾民; 沙杭; 高毅; 卢海

    2011-01-01

    目的 建立和鉴定人皮肤成纤维细胞(HSF)来源的诱导性多潜能干细胞(iPS)。方法利用逆转录病毒将Sox2、Klf4、Oct4、c-Myc基因导入原代HSF细胞,将其编程为iPS细胞。检测细胞内源、外源基因表达量,鉴定外源基因是否插入iPS细胞,分析细胞核型,细胞碱性磷酸酶染色和免疫荧光染色,体内分化畸胎瘤,体外分化拟胚体实验,对建立的iPS细胞进行鉴定。结果(1)iPS细胞形态类似于胚胎干细胞(ES)。(2)iPS细胞内源多能基因(Nanog、Oct4、Rex1、Sox2)表达量增高,外源编程基因(Sox2、Klf4、Oct4、c-Myc)表达量降低。(3)琼脂糖凝胶电泳实验证实外源基因插入iPS细胞核内。(4)iPS细胞核型正常,碱性磷酸酶活性增高,表达ES细胞特异性蛋白。(5)iPS细胞在体内能分化为畸胎瘤,在体外能分化为拟胚体。结论新建立的iPS细胞类似于ES细胞具有多向分化潜能。%Objective Generation of human induced pluripotent stem cells from human skin fibroblasts.Methods Sox2, Klf4, Oct4, c-Myc were transfected into HSF cells with retrovirus, and then HSF cells was reprogrammed to iPS cells.Detecting cells endogenous and exogenous gene, analyzing karyotype,cells alkaline phosphatase staining and immunofluorescence staining, differentiating into teratomas in vivo and embryoid bodies in vitro were performed.Results iPS cell morphology was similar to embryonic stem cells (ES).The expression of Nanog, Oct4, Rex1, Sox2 in iPS cells were higher than HSF cells, and Sox2, Klf4, Oct4, c-Myc were silenced for the iPS cells.Exogenous genes were inserted into the nucleus of iPS cells, which was confirmed by 1% agarose gel electrophoresis.iPS cell karyotype was normal, alkaline phosphatase activity increased, ES cell-specific protein expressed.iPS cells were differentiated into a teratoma in vivo and embryoid bodies in vitro.Conclusions iPS cells were similar to ES cells, which have pluripotency.

  1. Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

    Energy Technology Data Exchange (ETDEWEB)

    Tashiro, Kanae [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Shishido, Mayumi [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Fujimoto, Keiko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan); Hirota, Yuko [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Yo, Kazuyuki; Gomi, Takamasa [Skin Research Department, POLA Chemical Industries, Inc., Yokohama (Japan); Tanaka, Yoshitaka, E-mail: tanakay@bioc.phar.kyushu-u.ac.jp [Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka (Japan); Organelle Homeostasis Research Center, Kyushu University, Fukuoka (Japan)

    2014-01-03

    Highlights: •Autophagosomes accumulate in aged dermal fibroblasts. •Autophagic degradation is impaired in aged dermal fibroblasts. •Autophagy disruption affects extracellular matrix components in dermal fibroblasts. -- Abstract: Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.

  2. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    Directory of Open Access Journals (Sweden)

    Hansíková Hana

    2008-01-01

    Full Text Available Abstract Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate

  3. Skin Diseases: Skin Health and Skin Diseases

    Science.gov (United States)

    Skip Navigation Bar Home Current Issue Past Issues Skin Diseases Skin Health and Skin Diseases Past Issues / Fall 2008 Table of Contents ... acne to wrinkles Did you know that your skin is the largest organ of your body? It ...

  4. Advances in Skin Substitutes—Potential of Tissue Engineered Skin for Facilitating Anti-Fibrotic Healing

    Directory of Open Access Journals (Sweden)

    Mathew Varkey

    2015-07-01

    Full Text Available Skin protects the body from exogenous substances and functions as a barrier to fluid loss and trauma. The skin comprises of epidermal, dermal and hypodermal layers, which mainly contain keratinocytes, fibroblasts and adipocytes, respectively, typically embedded on extracellular matrix made up of glycosaminoglycans and fibrous proteins. When the integrity of skin is compromised due to injury as in burns the coverage of skin has to be restored to facilitate repair and regeneration. Skin substitutes are preferred for wound coverage when the loss of skin is extensive especially in the case of second or third degree burns. Different kinds of skin substitutes with different features are commercially available; they can be classified into acellular skin substitutes, those with cultured epidermal cells and no dermal components, those with only dermal components, and tissue engineered substitutes that contain both epidermal and dermal components. Typically, adult wounds heal by fibrosis. Most organs are affected by fibrosis, with chronic fibrotic diseases estimated to be a leading cause of morbidity and mortality. In the skin, fibroproliferative disorders such as hypertrophic scars and keloid formation cause cosmetic and functional problems. Dermal fibroblasts are understood to be heterogeneous; this may have implications on post-burn wound healing since studies have shown that superficial and deep dermal fibroblasts are anti-fibrotic and pro-fibrotic, respectively. Selective use of superficial dermal fibroblasts rather than the conventional heterogeneous dermal fibroblasts may prove beneficial for post-burn wound healing.

  5. Changes in skin levels of two neutotrophins (glial cell line derived neurotrohic factor and neurotrophin-3) cause alterations in cutaneous neuron responses to mechanical stimuli

    Institute of Scientific and Technical Information of China (English)

    Jeffrey Lawson; Sabrina L. Mcllwrath; H. Richard Koerber

    2008-01-01

    Neurotrophins are important for the development and maintenance of both high and low threshold mechanoreceptors (HTMRs and LTMRs). In this series of studies, the effects of constitutive overexpression of two different neurotrophins, neurotrophin-3 (NT-3) and glial cell line derived neurotrohic factor (GDNF), were examined. Previous studies indicated that both of them may be implicated in the normal development of mouse dorsal root ganglion (DRG) neurons. Neurons from mice transgenically altered to overexpress NT-3 or GDNF (NT-3-OE or GDNF-OE mice) in the skin were examined using several physiological, immunohistochemi-cal and molecular techniques. Ex vivo skin/nerve/DRG/spinal cord and skin/nerve preparations were used to determine the response characteristics of the cutaneous neurons; immunohistochemistry was used to examine the biochemical phenotype of DRG cells and the skin; RT-PCR was used to examine the levels of candidate ion channels in skin and DRG that may correlate with changes in physiologi-cal responses. In GDNF-OE mice, I-isolectin B4 (IB4)-immunopositive C-HTMRs (nociceptors), a large percentage of which are sensitive to GDNF, had significantly lower mechanical thresholds than wildtype (WT) neurons. Heat thresholds for the same cells were not different. Mechanical sensitivity changes in GDNF-OE mice were correlated with significant increases in acid sensing ion channels 2a (ASIC2a) and 2b (ASIC2b) and transient receptor potential channel AI (TRPAI), all of which are putative mechanosensitive ion channels. Overexpression of NT-3 affected the responses of A-LTMRs and A-HTMRs, hut had no effect on C-HTMRs. Slowly adapting type 1 (SA1) LTMRs and A-HTMRs had increased mechanical sensitivity compared to WT. Mechanical sensitivity was correlated with significant increases in acid-sensing ion channels ASIC1 and ASIC3. This data indicates that both neurotrophins play roles in determining mechanical thresholds of cutaneous HTMRs and LTMRs and that sensitivity

  6. Beryllium nitrate inhibits fibroblast migration to disrupt epimorphic regeneration.

    Science.gov (United States)

    Cook, Adam B; Seifert, Ashley W

    2016-10-01

    Epimorphic regeneration proceeds with or without formation of a blastema, as observed for the limb and skin, respectively. Inhibition of epimorphic regeneration provides a means to interrogate the cellular and molecular mechanisms that regulate it. In this study, we show that exposing amputated limbs to beryllium nitrate disrupts blastema formation and causes severe patterning defects in limb regeneration. In contrast, exposing full-thickness skin wounds to beryllium only causes a delay in skin regeneration. By transplanting full-thickness skin from ubiquitous GFP-expressing axolotls to wild-type hosts, we demonstrate that beryllium inhibits fibroblast migration during limb and skin regeneration in vivo Moreover, we show that beryllium also inhibits cell migration in vitro using axolotl and human fibroblasts. Interestingly, beryllium did not act as an immunostimulatory agent as it does in Anurans and mammals, nor did it affect keratinocyte migration, proliferation or re-epithelialization, suggesting that the effect of beryllium is cell type-specific. While we did not detect an increase in cell death during regeneration in response to beryllium, it did disrupt cell proliferation in mesenchymal cells. Taken together, our data show that normal blastema organogenesis cannot occur without timely infiltration of local fibroblasts and highlights the importance of positional information to instruct pattern formation during regeneration. In contrast, non-blastemal-based skin regeneration can occur despite early inhibition of fibroblast migration and cell proliferation.

  7. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  8. Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-gamma-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin.

    Science.gov (United States)

    Fromm, Sharon Vigodman; Duady-Ben Yaakov, Shirly; Schechter, Chana; Ehrlich, Rachel

    2002-02-01

    Antigen processing and presentation by class I MHC molecules generally require assembly with peptide epitopes generated by the proteasome and transported into the ER by the transporters associated with antigen presentation (TAP). Recently, TAP-independent pathways supporting class I MHC-mediated presentation of exogenous antigens, as well as of endogenously synthesized viral antigens, were described. We now characterize a TAP-independent pathway that is operative in both TAP1- and TAP2-deficient Adenovirus (Ad)-transformed fibroblast cell lines. To the best of our knowledge, this is the first time that the existence of such a pathway has been described in non-infected cells that do not belong to the hematopoietic lineage. We show that this pathway is proteasome-independent and chloroquine-sensitive. Cell surface expression of these TAP-independent class I complexes is modulated by tapasin levels and is enhanced by IFN-gamma. The data imply that IFN-gamma increases the relative level of TAP-independent high affinity class I complexes that exit the ER on their way to the cell surface and to vacuolar compartments where peptide cleavage/exchange might take place before recycling to the cell surface. Since both TAP and tapasin expression are altered in numerous tumors and in virus-infected cells, TAP-independent class I complexes may be a valuable target source for immune responses.

  9. Effects of glial cell line-derived neurotrophic factor, fibroblast growth factor 2 and epidermal growth factor on proliferation and the expression of some genes in buffalo (Bubalus bubalis) spermatogonial cells.

    Science.gov (United States)

    Kadam, Prashant H; Kala, Sushila; Agrawal, Himanshu; Singh, Karn P; Singh, Manoj K; Chauhan, Manmohan S; Palta, Prabhat; Singla, Suresh K; Manik, Radhay S

    2013-01-01

    The present study evaluated the effects of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor (FGF) 2 and epidermal growth factor (EGF) on proliferation and the expression of some genes in spermatogonial cells. Spermatogonial cells were isolated from prepubertal buffalo testes and enriched by double enzyme treatment, filtration through 80- and 60-μm nylon mesh filters, differential plating on lectin-coated dishes and Percoll density gradient centrifugation. Cells were then cultured on a buffalo Sertoli cell feeder layer and formed colonies within 15-18 days. The colonies were found to predominantly contain undifferentiated Type A spermatogonia because they bound Dolichos biflorus agglutinin and did not express c-kit. The colonies expressed alkaline phosphatase, NANOG, octamer-binding transcription factor (OCT)-4 and tumour rejection antigen (TRA)-1-60. Cells were subcultured for 15 days, with or without growth factor supplementation. After 15 days, colony area and the relative mRNA abundance of PLZF were higher (Pgrowth factor supplementation. In the Sertoli cell feeder layer, EGF and FGF2 decreased (Pgrowth factors was developed for the short-term culture of buffalo spermatogonia.

  10. Sarcophine-Diol, a Skin Cancer Chemopreventive Agent, Inhibits Proliferation and Stimulates Apoptosis in Mouse Melanoma B16F10 Cell Line

    Directory of Open Access Journals (Sweden)

    Hesham Fahmy

    2011-12-01

    Full Text Available Sarcodiol (SD is a semi-synthetic derivative of sarcophine, a marine natural product. In our previous work, we reported the significant chemopreventive effects of SD against non-melanoma skin cancer both in vitro and in vivo mouse models. In this investigation, we extended this work to study the effect of sarcodiol on melanoma development, the more deadly form of skin cancer, using the mouse melanoma B16F10 cell line. In this study we report that SD inhibits the de novo DNA synthesis and enhances fragmentation of DNA. We also evaluated the antitumor effect of SD on melanoma cell viability using several biomarkers for cell proliferation and apoptosis. SD inhibits the expression levels of signal transducers and activators of transcription protein (STAT-3 and cyclin D1, an activator of cyclin-dependent kinase 4 (Cdk4. SD treatment also enhances cellular level of tumor suppressor protein 53 (p53 and stimulates cleavage of the nuclear poly (ADP-ribose polymerase (cleaved-PARP. SD also enhances cellular levels of cleaved Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. These results, in addition to inhibition of cell viability, suggest that SD inhibits melanoma cell proliferation by arresting the cell-division cycle in a Go quiescent phase and activates programmed cell death (apoptosis via extrinsic and intrinsic pathways. Finally, these studies demonstrate that SD shows a very promising chemopreventive effect in melanoma B16F10 tumor cells.

  11. Sarcophine-diol, a skin cancer chemopreventive agent, inhibits proliferation and stimulates apoptosis in mouse melanoma B₁₆F₁₀ cell line.

    Science.gov (United States)

    Szymanski, Pawel T; Kuppast, Bhimanna; Ahmed, Safwat A; Khalifa, Sherief; Fahmy, Hesham

    2012-01-01

    Sarcodiol (SD) is a semi-synthetic derivative of sarcophine, a marine natural product. In our previous work, we reported the significant chemopreventive effects of SD against non-melanoma skin cancer both in vitro and in vivo mouse models. In this investigation, we extended this work to study the effect of sarcodiol on melanoma development, the more deadly form of skin cancer, using the mouse melanoma B₁₆F₁₀ cell line. In this study we report that SD inhibits the de novo DNA synthesis and enhances fragmentation of DNA. We also evaluated the antitumor effect of SD on melanoma cell viability using several biomarkers for cell proliferation and apoptosis. SD inhibits the expression levels of signal transducers and activators of transcription protein (STAT-3) and cyclin D1, an activator of cyclin-dependent kinase 4 (Cdk4). SD treatment also enhances cellular level of tumor suppressor protein 53 (p53) and stimulates cleavage of the nuclear poly (ADP-ribose) polymerase (cleaved-PARP). SD also enhances cellular levels of cleaved Caspase-3, -8, -9 and stimulates enzymatic activities of Caspase-3, -8 and -9. These results, in addition to inhibition of cell viability, suggest that SD inhibits melanoma cell proliferation by arresting the cell-division cycle in a Go quiescent phase and activates programmed cell death (apoptosis) via extrinsic and intrinsic pathways. Finally, these studies demonstrate that SD shows a very promising chemopreventive effect in melanoma B₁₆F₁₀ tumor cells.

  12. UVB诱导人皮肤成纤维细胞的氧化应激损伤研究%Oxidative stress in human skin fibroblasts induced by UVB irradiation

    Institute of Scientific and Technical Information of China (English)

    王懿娜; 吴炜; 彭国平; 方红

    2008-01-01

    目的 观察人皮肤成纤维细胞被UVB照射后的光损伤、凋亡、周期阻滞和氧化应激损伤状态,并检测氧化应激的相关信号蛋白p66Shc的表达情况.方法 用小剂量UVB多次照射培养的人皮肤成纤维细胞,以细胞衰老β-半乳糖苷酶(SA-β-Gal)化学染色法观察细胞衰老状态,流式细胞仪检测细胞凋亡和细胞周期,ELISA法检测细胞内超氧化物歧化酶活性和丙二醛的含量,并以Western印迹法检测p66Shc蛋白的表达.结果 SA-β-Gal染色显示,培养的人皮肤成纤维细胞在UVB照射后呈强阳性染色;细胞凋亡率在未照射的对照组为0.96%,UVB照射组为37%;而细胞周期检测显示,经UVB照射的人皮肤成纤维细胞大部分阻滞于G0/G1期(80.07%).细胞内超氧化物歧化酶水平对照组为(52.35±4.97)ng/g蛋白,UVB照射组为(7.8l±0.68)ng/g蛋白(两组比较,P<0.01);而丙二醛水平两组分别为(3.52±0.34)ng/g蛋白和(33.91±3.20)ng/g蛋白(两组比较,P<0.05).人皮肤成纤维细胞经UVB照射诱导后24 h,p66Shc呈弱阳性表达,48 h后表达进一步增强.结论 培养的人皮肤成纤维细胞经UVB照射后进人氧化应激增强状态.p66Shc蛋白在此过程中表达逐渐增强.%Objective To observe the aging,apoptosis,cell cycle arrest and oxidative stress in human skin fibroblast(HSF)induced by UVB,and to detect the expression profiles of p66Shc,a determinant of oxidative stress response and life span,in this process.Methods HSF cells were exposed to UVB at a subcytotoxic dosage twice a day for three days.The cells without exposure served as control.After another 24-hour culture,SA-β-Gal staining was performed to evaluate the senescence state of the cells,flow cytometry to observe cell apoptosis;cell cycle arrest was detected by serum starvation and flow cytometry:ELISA was applied to detect intracellular levels of superoxide dismutase(SOD)and malondialdehvde(MDA),and Western blotting to analyze the expression of p66

  13. In situ hybridization of the feline major satellite DNA FA-SAT in feline fibrosarcoma cell lines and feline fibrosarcoma tissue sections

    OpenAIRE

    Alfaro Alarcón, Alejandro

    2009-01-01

    Feline fibrosarcomas are the most common skin tumors of cats. Despite this high frequency and the publication of different hypotheses for their pathogenesis by several authors, the alterations accompanying the development of this tumor are still not completely understood. We studied the feline major satellite DNA (FA-SAT) hybridization pattern by FISH in four fibrosarcoma cell lines and one normal embryonic fibroblastic cell line as well as in 30 fibrosarcomas from 28 cats. Of the latter, ...

  14. Metallic copper nanoparticles induce apoptosis in a human skin melanoma A-375 cell line

    Science.gov (United States)

    Chakraborty, Ruchira; Basu, Tarakdas

    2017-03-01

    In two earlier communications (Chatterjee et al 2012 Nanotechnology 23 085103, Chatterjee et al 2014 Nanotechnology 25 135101), we reported the development of a simple and unique method of synthesizing highly stable metallic copper nanoparticles (Cu NPs) with high antibacterial activity. Here we report on the cytotoxic potency of the NPs against cancer cells. The value of the IC50 dose of the Cu NPs against human skin cancer cell A-375 was found to be 1.71 μg ml‑1 only, which was much less than values reported so far, and this concentration had no cytotoxic effect on normal white blood cells. The NPs caused (i) lowering of cell membrane rigidity, (ii) DNA degradation, (iii) chromosomal condensation, (iv) cell cycle arrest in the G2/M phase, (v) depolarization of the mitochondrial membrane and (vi) apoptosis of cells. Cellular apoptosis occurred in the caspase-9-mediated intrinsic pathway. This study revealed that our Cu NPs had high anticancer properties by killing tumor cells through the apoptotic pathway. Since this particle has high antibacterial activity, our Cu NPs might be developed in future as a dual action drug—anticancer as well as antibacterial.

  15. κ-卡拉胶寡糖对小鼠皮肤成纤维细胞增殖和胶原分泌的影响%Effects of κ-carrageenan oligosaccharides on cell proliferation and collagen secretion in cultured mouse skin fibroblast

    Institute of Scientific and Technical Information of China (English)

    王天琦; 吴海歌; 姚子昂

    2013-01-01

    目的研究κ-卡拉胶寡糖对小鼠皮肤成纤维细胞增殖和胶原分泌的影响.方法原代培养小鼠皮肤成纤维细胞,传代培养后加入不同浓度的κ-卡拉胶寡糖作用,应用细胞计数法,四甲基偶氮唑盐(MTT)比色法,检测κ--卡拉胶寡糖对皮肤成纤维细胞增殖的影响,ELISA法检测κ--卡拉胶寡糖对皮肤成纤维细胞分泌Ⅰ型、Ⅲ型胶原蛋白的影响.结果12.5μg/mL~100μg,/mL范围内,各组κ-卡拉胶寡糖对小鼠皮肤成纤维细胞均有一定促进增殖作用,其中以100μg/mL时最为显著(P<0.01).用25μg/mL和50μg/mLκ-卡拉胶寡糖处理小鼠皮肤成纤维细胞后,与对照组相比,Ⅰ型胶原蛋白分泌明显增加(P<0.05);12.5μg/mL~100μg/mL范围内,各组κ-卡拉胶寡糖均显著促进Ⅲ型胶原蛋白分泌(P<0.05).结论κ-卡拉胶寡糖能有效促进小鼠皮肤成纤维细胞增殖和胶原分泌.%Objective: To investigate the effect of κ - carrageenan oligosaccharides on cell proliferation and collagen secretion in cultured mouse skin fibroblasts. Methods; The primary mouse skin fibroblasts were isolated and cultured in vitro. The cells were treated with κ- carrageenan oligosaccharides in different concentration in exponential phase of cell growth. The cell proliferation was measured by cell counting and MTT colormetric assay. Type I and type IE collagen secretion was detected by ELJSA. Results: Compared with the control group,the growth of mouse skin fibroblasts was enhanced. When the cells were stimulated by κ- carrageenan oligosaccharides,100μg/mL is most significant (P < 0.01). Type Ⅰ collagen secretion was increased (P<0.05) after the cells were stimulated by 25μg/mL and 50μg/mL κ - carrageenan oligosaccharides; type Ⅲ collagen secretion was increased ( P < 0. 05) after the cells were stimulated by k - carrageenan oligosaccharides. Conclusion: κ - carrageenan oligosaccharides can enhance the growth of mouse skin fibroblasts and

  16. Fibroblastic rheumatism: case report and review of the literature.

    Science.gov (United States)

    Lee, Joyce M; Sundel, Robert P; Liang, Marilyn G

    2002-01-01

    A previously healthy 7-year-old boy presented with polyarthritis and skin lesions. Multiple, skin- to pink-colored, firm papules were noted on the periungual areas, hands, feet, and nose. There was swelling of the proximal interphalangeal joints, wrists, elbows, ankles, and knees. A skin biopsy specimen revealed dermal fibrosis and interspersed histiocytes and lymphocytes. These findings were consistent with fibroblastic rheumatism, a condition characterized by cutaneous nodules and a symmetric polyarthritis. He was treated with methotrexate and corticosteroids with improvement in the symptoms of his arthritis and skin lesions. This early treatment was beneficial in our patient.

  17. Rac inhibition reverses the phenotype of fibrotic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Shi-wen Xu

    Full Text Available BACKGROUND: Fibrosis, the excessive deposition of scar tissue by fibroblasts, is one of the largest groups of diseases for which there is no therapy. Fibroblasts from lesional areas of scleroderma patients possess elevated abilities to contract matrix and produce alpha-smooth muscle actin (alpha-SMA, type I collagen and CCN2 (connective tissue growth factor, CTGF. The basis for this phenomenon is poorly understood, and is a necessary prerequisite for developing novel, rational anti-fibrotic strategies. METHODS AND FINDINGS: Compared to healthy skin fibroblasts, dermal fibroblasts cultured from lesional areas of scleroderma (SSc patients possess elevated Rac activity. NSC23766, a Rac inhibitor, suppressed the persistent fibrotic phenotype of lesional SSc fibroblasts. NSC23766 caused a decrease in migration on and contraction of matrix, and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. SSc fibroblasts possessed elevated Akt phosphorylation, which was also blocked by NSC23766. Overexpression of rac1 in normal fibroblasts induced matrix contraction and alpha-SMA, type I collagen and CCN2 mRNA and protein expression. Rac1 activity was blocked by PI3kinase/Akt inhibition. Basal fibroblast activity was not affected by NSC23766. CONCLUSION: Rac inhibition may be considered as a novel treatment for the fibrosis observed in SSc.

  18. A first vascularized skin equivalent for as an alternative to animal experimentation.

    Science.gov (United States)

    Groeber, Florian; Engelhardt, Lisa; Lange, Julia; Kurdyn, Szymon; Schmid, Freia F; Rücker, Christoph; Mielke, Stephan; Walles, Heike; Hansmann, Jan

    2016-01-01

    Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.

  19. Lines of Blaschko.

    Science.gov (United States)

    Bolognia, J L; Orlow, S J; Glick, S A

    1994-08-01

    The lines of Blaschko represent a pattern followed by many skin disorders. We review the clinical and histologic features of X-linked, congenital/nevoid, and acquired skin diseases that follow these lines. We also include cutaneous disorders that have a linear distribution but do not follow Blaschko's lines. Finally, we differentiate Blaschko's lines from other patterns on the skin such as dermatomes and Langer's lines.

  20. Your Skin

    Science.gov (United States)

    ... Room? What Happens in the Operating Room? Your Skin KidsHealth > For Kids > Your Skin Print A A ... are really dead skin cells. continue Bye-Bye Skin Cells These old cells are tough and strong, ...

  1. Evaluation of cytotoxic activities of snake venoms toward breast (MCF-7) and skin cancer (A-375) cell lines.

    Science.gov (United States)

    Bradshaw, Michael J; Saviola, Anthony J; Fesler, Elizabeth; Mackessy, Stephen P

    2016-08-01

    Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeutics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and "Colubridae") in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells. MTT cell viability assays of cancer cells incubated with crude venoms revealed that most venoms showed significant cytotoxicity. We further investigated venom from the Red-bellied Blacksnake (Pseudechis porphyriacus); venom was fractionated by ion exchange fast protein liquid chromatography and several cytotoxic components were isolated. SDS-PAGE and MALDI-TOF mass spectrometry were used to identify the compounds in this venom responsible for the cytotoxic effects. In general, viper venoms were potently cytotoxic, with MCF-7 cells showing greater sensitivity, while elapid and colubrid venoms were much less toxic; notable exceptions included the elapid genera Micrurus, Naja and Pseudechis, which were quite cytotoxic to both cell lines. However, venoms with the most potent cytotoxicity were often not those with low mouse LD50s, including some dangerously venomous viperids and Australian elapids. This study confirmed that many venoms contain cytotoxic compounds, including catalytic PLA2s, and several venoms also showed significant differential toxicity toward the two cancer cell lines. Our results indicate that several previously uncharacterized venoms could contain promising lead compounds for drug development.

  2. 长波紫外线对人皮肤成纤维细胞自噬水平的影响%Effects of ultraviolet A on autophagy in human skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    郑云鹏; 陈旭; 黄丹; 徐松; 顾恒

    2015-01-01

    Objective To evaluate the effects of ultraviolet A (UVA) on autophagy in human skin fibroblasts (HSFs).Methods Cultured HSFs were randomly divided into chronic and acute UVA radiation groups.HSFs in the chronic UVA radiation groups were irradiated with UVA at 5,10 and 20 J/cm2 separately once a day for 4 consecutive days,with HSFs receiving no radiation serving as the chronic radiation control group;HSFs in the acute UVA radiation groups received a single session of radiation with 5,10,30 and 60 J/cm2 UVA separately,with HSFs receiving no radiation serving as the acute radiation control group.After additional culture for different durations,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of HSFs,monodansylcadaverin (MDC) staining to determine autophagy levels,and Western blot analysis to track the conversion of the microtubule-associated protein 1 light chain-3 (LC3)-Ⅰ to LC3-Ⅱ.Statistical analysis was carried out by using one-way analysis of variance followed by Students-Newman-Keuls (SNK) test for multiple-group comparisons and by the independent sample t test for two-group comparisons.Results The cellular proliferative activity significantly decreased in the 3 chronic radiation groups at 1 hour after the final UVA radiation compared with the chronic radiation control group (F =155.5,P < 0.05),and in the 4 acute radiation groups at 1,6 and 12 hours after UVA radiation compared with the acute radiation control group (F =1 335,1 649,2 774,all P < 0.05).MDC staining showed that the autophagy levels in HSFs significantly increased in the 3 chronic radiation groups after UVA radiation compared with the chronic radiation control group (F =748.62,P > 0.05),but showed no significant changes in any of the acute radiation groups at 1,6 or 12 hours after UVA radiation compared with the acute radiation control group (F =0.014,0.004,0.002,all P > 0.05).The ratio of LC3-Ⅱ to LC3-Ⅰ was significantly elevated in all

  3. The 'beauty' of skin neurobiology.

    Science.gov (United States)

    Pincelli, C; Bonté, F

    2003-07-01

    The skin is the most densely innervated organ in the body and there is a close relationship between the skin and the nervous system. Most skin cells express receptors for neuromediators (NM) and skin cells themselves are an important source of NM. In particular, human keratinocytes synthesize neurotrophins and endorphins and express their receptors. In addition to neurotrophic activity, NM are involved in skin homeostasis, trophism and stress responses. NM released from keratinocytes also function in a paracrine fashion on other skin cells, such as Langerhans cells, melanocytes and fibroblasts. We discuss the influence of NM on these cells, which may be involved in major cosmetic problems like ageing, baldness and dyspigmentation. Based on this correlation, it seems reasonable to target neural factors for cosmetic purposes.

  4. Anti-skin cancer properties of phenolic-rich extract from the pericarp of mangosteen (Garcinia mangostana Linn.).

    Science.gov (United States)

    Wang, Jing J; Shi, Qing H; Zhang, Wei; Sanderson, Barbara J S

    2012-09-01

    Skin cancers are often resistant to conventional chemotherapy. This study examined the anti-skin cancer properties of crude ethanol extract of mangosteen pericarp (MPEE) on human squamous cell carcinoma A-431 and melanoma SK-MEL-28 lines. Significant dose-dependent reduction in% viability was observed for these cell lines, with less effect on human normal skin fibroblast CCD-1064Sk and keratinocyte HaCaT cell lines. Cell distribution in G(1) phase (93%) significantly increased after 10 μg/ml of MPEE versus untreated SK-MEL-28 cells (78%), which was associated with enhanced p21(WAF1) mRNA levels. In A-431 cells, 10 μg/ml MPEE significantly increased the sub G(1) peak (15%) with concomitant decrease in G(1) phase over untreated cells (2%). In A-431 cells, 10 μg/ml MPEE induced an 18% increase in early apoptosis versus untreated cells (2%). This was via caspase activation (15-, 3- and 4-fold increased caspse-3/7, 8, and 9 activities), and disruption of mitochondrial pathways (6-fold decreased mitochondrial membrane potential versus untreated cells). Real-time PCR revealed increased Bax/Bcl-2 ratio and cytochrome c release, and decreased Akt1. Apoptosis was significantly increased after MPEE treatment of SK-MEL-28 cells. Hence, MPEE showed strong anti-skin cancer effect on these two skin cancer cell lines, with potential as an anti-skin cancer agent.

  5. 含鼠胚胎成纤维细胞的组织工程皮肤体外构建及大鼠移植研究%Construction and transplantation of tissue-engineered skin with mouse embryonic fibroblasts in SD mice

    Institute of Scientific and Technical Information of China (English)

    刘柳; 李武德; 蔡国斌

    2011-01-01

    Objective To investigate the application and mechanism of tissue-engineered skin with mouse embryonic fibroblasts(MEFs)for the full-thickness skin defects on mice.Methods The MEFs and fibroblasts were cultured and seeded in scaffold made of rat tail collage.ELISA method was used for detection of secretory function.The full-thickness skin defects were created on mice and covered by MEFsscaffold complex(experimental group),or FBs-scaffold complex(control group 1),or scaffold only(control group 2).The process of wound healing was evaluated by observation of the re-epithelization rate.Microvessal density(MVD)and vimentin within the wound sites were also detected with immunohistochemistry staining technique to describe the characteristics of wound healing.Hoechst 33342 staining was performed to trace MEFs'fate.Results MEFs scaffold group had higher level secretion of IL-6 and lower of TGF-β1 than FBs scaffold group(P<0.05).Compared with wounds in control groups,the wounds in MEFs group healed markedly fast(P<0.05)and the MVD was significantly higher(P<0.05).The fibroblasts in the wounds of MEFs group were arranged regularly and the MEFs decreased during the healing process.Conclusions The MEFs-scaffold complex can promote wound healing with less scar formation.MEFs may have an inducing effect on the wound healing.%目的 利用鼠胚胎成纤维细胞(mouse embryonic fibrobalsts,MEFs)三维培养修复大鼠全层皮肤缺损,并初步探讨其机制.方法 培养MEFs和普通成纤维细胞(fibroblasts,FBs),复合鼠尾胶原形成三维构建,ELASA法测定其分泌功能.制作大鼠全层皮肤缺损,按移植物不同分为3组:MEFs组(实验组)、FBs组(对照组1)和空白胶原组(对照组2).观察愈合时间并计算愈合面积比率,定期取创面组织行CD31、波形蛋白免疫组化检测,并以Hoechst33342荧光标记MEFs,示踪其转归.结果 与FBs组相比,MEFs组的IL-6分泌更加旺盛,而TGF-β1分泌量少(P<0.05).

  6. Differential Expression of Matrix Metalloproteases in Human Fibroblasts with Different Origins

    Directory of Open Access Journals (Sweden)

    Diana Lindner

    2012-01-01

    Full Text Available Fibroblasts are widely distributed cells and are responsible for the deposition of extracellular matrix (ECM components but also secrete ECM-degrading matrix metalloproteases. A finely balanced equilibrium between deposition and degradation of ECM is essential for structural integrity of tissues. In the past, fibroblasts have typically been understood as a uniform cell population with comparable functions regardless of their origin. Here, we determined growth curves of fibroblasts derived from heart, skin, and lung and clearly show the lowest proliferation rate for cardiac fibroblasts. Furthermore, we examined basal expression levels of collagen and different MMPs in these three types of fibroblasts and compared these concerning their site of origin. Interestingly, we found major differences in basal mRNA expression especially for MMP1 and MMP3. Moreover, we treated fibroblasts with TNF-α and observed different alterations under these proinflammatory conditions. In conclusion, fibroblasts show different properties in proliferation and MMP expression regarding their originated tissue.

  7. Dysregulated proinflammatory and fibrogenic phenotype of fibroblasts in cystic fibrosis.

    Directory of Open Access Journals (Sweden)

    François Huaux

    Full Text Available Morbi-mortality in cystic fibrosis (CF is mainly related to chronic lung infection and inflammation, uncontrolled tissue rearrangements and fibrosis, and yet the underlying mechanisms remain largely unknown. We evaluated inflammatory and fibrosis responses to bleomycin in F508del homozygous and wild-type mice, and phenotype of fibroblasts explanted from mouse lungs and skin. The effect of vardenafil, a cGMP-specific phosphodiesterase type 5 inhibitor, was tested in vivo and in culture. Responses of proinflammatory and fibrotic markers to bleomycin were enhanced in lungs and skin of CF mice and were prevented by treatment with vardenafil. Purified lung and skin fibroblasts from CF mice proliferated and differentiated into myofibroblasts more prominently and displayed higher sensitivity to growth factors than those recovered from wild-type littermates. Under inflammatory stimulation, mRNA and protein expression of proinflammatory mediators were higher in CF than in wild-type fibroblasts, in which CFTR expression reached similar levels to those observed in other non-epithelial cells, such as macrophages. Increased proinflammatory responses in CF fibroblasts were reduced by half with submicromolar concentrations of vardenafil. Proinflammatory and fibrogenic functions of fibroblasts are upregulated in CF and are reduced by vardenafil. This study provides compelling new support for targeting cGMP signaling pathway in CF pharmacotherapy.

  8. The Effect of PRP Promoting the Expression of Collagen and Hyaluronic Acid of Light Aging Human Skin Fibroblasts in Vitro%PRP促进光老化人皮肤成纤维细胞胶原与透明质酸合成的研究

    Institute of Scientific and Technical Information of China (English)

    邓辰亮; 常毅; 万伟东; 郑江红; 屈悦; 茅广宇; 丁志; 杨松林

    2012-01-01

    Objective To explore the effects of platelet-rich plasma (PRP) on collagen and hyaluronic acid (HA) expression of light aging human skin fibroblasts (HSFs) in vitro. Methods Cultured human skin fibroblasts were radiated with ultraviolet ray and the proliferation and doubling time of cell were estimated. Different concentrations (10%, 30% and 50%) of PRP were added into cultured light aging human skin fibroblasts, and HSFs radiated with ultraviolet ray were taken as negative control and HSFs were taken as positive control. ELISA was used to measure collagen expression and hyaluronic acid expression. Results The cell proliferation was prohibited after ultraviolet irradiation and cell doubling time was shortened with the increasing of PRP concentration after PRP added. ELISA assay showed that the collagen type 1 (COL1), type 3 (COL3) and HA expression were decreased after ultraviolet irradiation and promoted after PRP added, significant difference was shown between expermental groups and negative control group (P<0.01). Conclusion PRP can promote the synthesis of collagen type 1 (COL1), type 3 (COL3) and HA, and may be used as an effective factor against skin photoaging.%目的 体外研究人皮肤成纤维细胞(HSFs)发生光老化时,富血小板血浆(PRP)对其合成胶原与透明质酸的影响.方法 使用紫外线(UVA)照射体外培养的原代人皮肤成纤维细胞,并检测光老化细胞的细胞增殖情况及细胞倍增时间.用含不同浓度PRP(10%、30%、50%)的培养液培养光老化细胞,正常培养的光老化细胞为阴性对照,原代人皮肤成纤维细胞为阳性对照.ELISA检测各组细胞外液中胶原蛋白与透明质酸含量.结果 经紫外线照射后,细胞增殖减弱,加入PRP后,伴随PRP浓度的增加,细胞倍增时间缩短;ELISA检测发现,经过UVA照射的成纤维细胞COL1、COL3及透明质酸合成量,显著低于正常培养的成纤维细胞(P<0.01).在PRP作用后,成纤维细胞的COL1、COL3

  9. Abscisic acid ameliorates the systemic sclerosis fibroblast phenotype in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bruzzone, Santina, E-mail: santina.bruzzone@unige.it [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Battaglia, Florinda [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Mannino, Elena [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Parodi, Alessia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Fruscione, Floriana [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Advanced Biotechnology Center, Largo Rosanna Benzi 10, 16132 Genova (Italy); Basile, Giovanna [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Salis, Annalisa; Sturla, Laura [Department of Experimental Medicine, Section of Biochemistry, University of Genova, Viale Benedetto XV 1, 16132 Genova (Italy); Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Negrini, Simone; Kalli, Francesca; Stringara, Silvia [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Filaci, Gilberto [Centre of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV 9, 16132 Genova (Italy); Department of Internal Medicine, Viale Benedetto XV 6, 16132 Genova (Italy); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer ABA is an endogenous hormone in humans, regulating different cell responses. Black-Right-Pointing-Pointer ABA reverts some of the functions altered in SSc fibroblasts to a normal phenotype. Black-Right-Pointing-Pointer UV-B irradiation increases ABA content in SSc cultures. Black-Right-Pointing-Pointer SSc fibroblasts could benefit from exposure to ABA and/or to UV-B. -- Abstract: The phytohormone abscisic acid (ABA) has been recently identified as an endogenous hormone in humans, regulating different cell functions, including inflammatory processes, insulin release and glucose uptake. Systemic sclerosis (SSc) is a chronic inflammatory disease resulting in fibrosis of skin and internal organs. In this study, we investigated the effect of exogenous ABA on fibroblasts obtained from healthy subjects and from SSc patients. Migration of control fibroblasts induced by ABA was comparable to that induced by transforming growth factor-{beta} (TGF-{beta}). Conversely, migration toward ABA, but not toward TGF-{beta}, was impaired in SSc fibroblasts. In addition, ABA increased cell proliferation in fibroblasts from SSc patients, but not from healthy subjects. Most importantly, presence of ABA significantly decreased collagen deposition by SSc fibroblasts, at the same time increasing matrix metalloproteinase-1 activity and decreasing the expression level of tissue inhibitor of metalloproteinase (TIMP-1). Thus, exogenously added ABA appeared to revert some of the functions altered in SSc fibroblasts to a normal phenotype. Interestingly, ABA levels in plasma from SSc patients were found to be significantly lower than in healthy subjects. UV-B irradiation induced an almost 3-fold increase in ABA content in SSc cultures. Altogether, these results suggest that the fibrotic skin lesions in SSc patients could benefit from exposure to high(er) ABA levels.

  10. Polycomponent mesotherapy formulations for the treatment of skin aging and improvement of skin quality

    Directory of Open Access Journals (Sweden)

    Prikhnenko S

    2015-04-01

    Full Text Available Sergey Prikhnenko Private Practice, Novosibirsk, Russia Abstract: Skin aging can largely be attributed to dermal fibroblast dysfunction and a decrease in their biosynthetic activity. Regardless of the underlying causes, aging fibroblasts begin to produce elements of the extracellular matrix in amounts that are insufficient to maintain the youthful appearance of skin. The goal of mesopreparations is primarily to slow down and correct changes in skin due to aging. The rationale for developing complex polycomponent mesopreparations is based on the principle that aging skin needs to be supplied with the various substrates that are key to the adequate functioning of the fibroblast. The quintessential example of a polycomponent formulation – NCTF® (New Cellular Treatment Factor – includes vitamins, minerals, amino acids, nucleotides, coenzymes and antioxidants, as well as hyaluronic acid, designed to help fibroblasts function more efficiently by providing a more optimal environment for biochemical processes and energy generation, as well as resisting the effects of oxidative stress. In vitro experiments suggest that there is a significant increase in the synthetic and prophylactic activity of fibroblasts with treated NCTF, and a significant increase in the ability of cells to resist oxidative stress. The current article looks at the rationale behind the development of polycomponent mesopreparations, using NCTF as an example. Keywords: mesotherapy, skin aging, skin quality

  11. Immortalization of Werner syndrome and progeria fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H.; Moses, R.E. (Baylor College of Medicine, Houston, TX (USA))

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  12. Development of an in vitro skin sensitization test using human cell lines: the human Cell Line Activation Test (h-CLAT). I. Optimization of the h-CLAT protocol.

    Science.gov (United States)

    Ashikaga, T; Yoshida, Y; Hirota, M; Yoneyama, K; Itagaki, H; Sakaguchi, H; Miyazawa, M; Ito, Y; Suzuki, H; Toyoda, H

    2006-08-01

    The aim of this study is to optimize the experimental conditions for an in vitro skin sensitization test using the human cell lines THP-1 and U-937. As regards pre-culturing time, the expression of CD86 on DNCB-treated THP-1 cells tended to be higher after 48h and 72h pre-culture compared with other time points evaluated. Next, we investigated the effect of chemical treatment time, and found that induction of CD86 expression on THP-1 cells by DNCB reached a plateau after 24h. Augmentation of CD86 expression is often observed when cells are treated with a subtoxic dose of allergens. To determine the appropriate dose of test samples, the cytotoxicity of test samples to THP-1 and U-937 cells was assessed with MTT assay, and the 50% inhibitory concentration (IC50) of each test sample was calculated. Based on the cytotoxicity assay data, four concentrations in the range between toxic and non-toxic were selected (0.1x, 0.5x, 1x and 2x IC50). Several kinds of antibodies were tested for staining THP-1 and U-937 cells treated with allergens/non-allergens (e.g., DNCB, Ni/SLS), and suitable antibodies for staining CD86 and CD54 were selected. We confirmed that the working dilutions of the selected CD86 and CD54 antibodies were appropriate for use in our method. The effect of an FcR blocking procedure was also evaluated. The mean fluorescence intensity (MFI value) was decreased by the FcR blocking procedure, which indicated that non-specific staining was blocked. Therefore, this procedure should be included in the method. Based on our findings, the protocol for this assay was optimized and the experimental conditions to be used in a future validation study were identified. We propose to call this kind of in vitro skin sensitization test h-CLAT, which is short for human Cell Line Activation Test.

  13. Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Yira Bermudez

    Full Text Available BACKGROUND: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. RESULTS: Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. CONCLUSIONS: The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s of nicotinic acid receptors in human skin homeostasis.

  14. Dupuytren's Contracture: Fibroblast Contraction?

    Science.gov (United States)

    Gabbiani, Giulio; Majno, Guido

    1972-01-01

    In 6 cases of Dupuytren's disease and 1 of Ledderhose's disease, the nodules of the palmar and plantar aponeurosis were examined by light and electron microscopy. The cells composing these nodules, presumably fibroblasts, showed three significant ultrastructural features: (1) a fibrillar system similar to that of smooth muscle cells; (2) nuclear deformations such as are found in contracted cells, the severest being recognizable by light microscopy (cross-banded nuclei); (3) cell-to-cell and cell-to-stroma attachments. Based on these data and on recent information about the biology of the fibroblasts, it is suggested that these cells are fibroblasts that have modulated into contractile cells (myofibroblasts), and that their contraction plays a role in the pathogenesis of the contracture observed clinically. ImagesFig 10Fig 5Fig 11Fig 6 and 7Fig 8Fig 1Fig 2Fig 9Fig 3Fig 4 PMID:5009249

  15. Accelerated Telomere Shortening and Replicative Senescence in Human Fibroblasts Overexpressing Mutant and Wild Type Lamin A

    Science.gov (United States)

    Huang, Shurong; Risques, Rosa Ana; Martin, George M.; Rabinovitch, Peter S.; Oshima, Junko

    2008-01-01

    LMNA mutations are responsible for a variety of genetic disorders, including muscular dystrophy, lipodystrophy, and certain progeroid syndromes, notably Hutchinson-Gilford Progeria. Although a number of clinical features of these disorders are suggestive of accelerated aging, it is not known whether cells derived from these patients exhibit cellular phenotypes associated with accelerated aging. We examined a series of isogenic skin fibroblast lines transfected with LMNA constructs bearing known pathogenic point mutations or deletion mutations found in progeroid syndromes. Fibroblasts overexpressing mutant lamin A exhibited accelerated rates of loss of telomeres and shortened replicative lifespans, in addition to abnormal nuclear morphology. To our surprise, these abnormalities were also observed in lines overexpressing wild-type lamin A. Copy number variants are common in human populations; those involving LMNA, whether arising meiotically or mitotically, might lead to progeroid phenotypes. In an initial pilot study of 23 progeroid cases without detectible WRN or LMNA mutations, however, no cases of altered LMNA copy number were detected. Nevertheless, our findings raise a hypothesis that changes in lamina organization may cause accelerated telomere attrition, with different kinetics for overexpession of wild-type and mutant lamin A, which leads to rapid replicative senescence and progroid phenotypes. PMID:17870066

  16. An altered redox balance and increased genetic instability characterize primary fibroblasts derived from xeroderma pigmentosum group A patients

    Energy Technology Data Exchange (ETDEWEB)

    Parlanti, Eleonora [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Pietraforte, Donatella; Iorio, Egidio; Visentin, Sergio; De Nuccio, Chiara [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Zijno, Andrea; D’Errico, Mariarosaria; Simonelli, Valeria [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Sanchez, Massimo [Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Fattibene, Paola [Department of Technology and Health, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Falchi, Mario [National AIDS Center, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy); Dogliotti, Eugenia, E-mail: dogliotti@iss.it [Department of Environment and Primary Prevention, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome (Italy)

    2015-12-15

    Highlights: • Increased levels and different types of intracellular radical species as well as an altered glutathione redox state characterize XP-A human cells when compared to normal. • A more glycolytic metabolism and higher ATP levels are associated with alteration of mitochondrial morphology and response to mitochondrial toxicants when XPA is defective. • XP-A human cells show increased spontaneous micronuclei frequency, a hallmark of cancer risk. - Abstract: Xeroderma pigmentosum (XP)-A patients are characterized by increased solar skin carcinogenesis and present also neurodegeneration. XPA deficiency is associated with defective nucleotide excision repair (NER) and increased basal levels of oxidatively induced DNA damage. In this study we search for the origin of increased levels of oxidatively generated DNA lesions in XP-A cell genome and then address the question of whether increased oxidative stress might drive genetic instability. We show that XP-A human primary fibroblasts present increased levels and different types of intracellular reactive oxygen species (ROS) as compared to normal fibroblasts, with O{sub 2−}· and H{sub 2}O{sub 2} being the major reactive species. Moreover, XP-A cells are characterized by decreased reduced glutathione (GSH)/oxidized glutathione (GSSG) ratios as compared to normal fibroblasts. The significant increase of ROS levels and the alteration of the glutathione redox state following silencing of XPA confirmed the causal relationship between a functional XPA and the control of redox balance. Proton nuclear magnetic resonance ({sup 1}H NMR) analysis of the metabolic profile revealed a more glycolytic metabolism and higher ATP levels in XP-A than in normal primary fibroblasts. This perturbation of bioenergetics is associated with different morphology and response of mitochondria to targeted toxicants. In line with cancer susceptibility, XP-A primary fibroblasts showed increased spontaneous micronuclei (MN) frequency, a

  17. Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

    Science.gov (United States)

    Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

    2016-01-01

    Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

  18. Molecularly characterized solvent extracts and saponins from Polygonum hydropiper L show high anti-angiogenic, anti-tumor, brine shrimp and fibroblast NIH/3T3 cell line cytotoxicity

    Directory of Open Access Journals (Sweden)

    Muhammad eAyaz

    2016-03-01

    Full Text Available Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, GC-MS to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr, its subsequent fractions; n-hexane (Ph.Hex, chloroform (Ph.Chf, ethyl acetate (Ph.EtAc, n-Butanol (Ph.Bt, aqueous (Ph.Aq, saponins (Ph.Sp were performed using the chick embryo chorioallantoic membrane (CAM assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed on Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line using brine shrimps and MTT cells viability assays. The GC-MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt and Ph.EtAc identified 126, 124, 153, 131 and 164 compounds respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75 and 461.53 µg/ml respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19 and 342.53 µg/ml respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50 and 71.50% cytotoxicity respectively at 1000 µg/ml with the LD50 of 140, 160 and 175 µg/ml respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer.

  19. Skin Conditions

    Science.gov (United States)

    Your skin is your body's largest organ. It covers and protects your body. Your skin Holds body fluids in, preventing dehydration Keeps harmful ... it Anything that irritates, clogs, or inflames your skin can cause symptoms such as redness, swelling, burning, ...

  20. Microarray Analysis of Host Cell Gene Transcription in Response to Varicella-Zoster Virus Infection of Human T Cells and Fibroblasts In Vitro and SCIDhu Skin Xenografts In Vivo

    OpenAIRE

    Jones, Jeremy O.; Arvin, Ann M.

    2003-01-01

    During primary infection, varicella-zoster virus (VZV) is spread via lymphocytes to skin, where it induces a rash and establishes latency in sensory ganglia. A live, attenuated varicella vaccine (vOka) was generated by using the VZV Oka strain (pOka), but the molecular basis for vOka attenuation remains unknown. Little is known concerning the effects of wild-type or attenuated VZV on cellular gene regulation in the host cells that are critical for pathogenesis. In this study, transcriptional ...

  1. [Dynamics of local expression of connexin-43 and basic fibroblast growth factor receptors in patients with skin and soft-tissue infections against the background of diabetes mellitus type II].

    Science.gov (United States)

    Vinnik, Iu S; Salmina, A B; Tepliakova, O V; Drobushevskaia, A I; Malinovskaia, N A; Pozhilenkova, E A; Morgun, A V; Gitlina, A G

    2014-01-01

    Clinical results of wound healing dynamics were studied in 60 patients with soft-tissue infection against the background of diabetes mellitus type II. At the same time the study considered indices of intercellular contacts protein tissue expression such as connexin 43 (Cx43) and basic fibroblast growth factor receptors (bFGFR). The basic therapy of biopsy material of wound borders was applied. The reduction of bFGFR expression and the minor growth of Cx43 expression were observed. The pain syndrome proceeded for a long time and there were signs of perifocal inflammation, retard wound healing with granulation tissue. The application of combined method of ozone therapy which included autohemotherapy with ozone and an external management of wound by ozone-oxygen mixture facilitated to considerable shortening of inflammatory phase and regeneration. It was associated with increased Cx43 expression (in 1.9 times) in comparison with initial level and bFGFR was enlarged in 1.7 times to eighth day of postoperative period.

  2. 黄芪多糖对过氧化氢刺激皮肤成纤维细胞中线粒体和溶酶体的保护作用%Protective effect of astragalus polysaccharide on mitochondria and lysosome in H2O2 -stressed skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    岳晓莉; 李萍; 刘欣; 黄启福; BARBIER-CHASSEFIERE Véronique; MORIN Christophe; PAPY-GARCIA Dulce

    2008-01-01

    AIM: To verify the protection of astragalus polysaecharide (APS) on H2O2 - stressed skin fibro-blasts. METHODS: A model of acute H2O2 stress in primary skin fibroblast was used at concentration of 0.5 mmol/L by 30 rain incubation. Dose responses of APS on cell survival was measured by MTI, cell death was evaluated by DAPI, and effect of APS on mitochondria, mitochondrial membrane potential and lysesome stabilization were measured by confocal microscopy. RESULTS: APS improved cell survival in a dose -dependent manner, starting at 0. 5 mg/L and with a maximum at 1 mg/L. Moreover, APS inhibited mitochondrial membrane potential collapse, protected mitechondrial morphology and stablized lysosomal membrane. CONCLUSION: The results suggest the existence, at the mitochondria-lysosome level, of a new pathway of apoptotic regulation by APS. This might constitute a new therapeutic target where oxidative stress and lysesomal impairment are involved.%目的:证明黄芪多糖对氧化应激皮肤细胞的保护作用.方法:原代培养皮肤成纤维细胞在过氧化氢0.5 mmol/L中.孵育30 min造成急性氧化损伤的模型.MTY法测黄芪多糖的剂量反应,DAPI染色法检测黄芪多糖对细胞死亡的作用;激光共聚焦法检测黄芪多糖对线粒体、线粒体膜电位以及溶酶体稳定性的保护作用.结果:经过黄芪多糖的治疗,能够明显提高细胞存活率,其作用浓度从0.5 ms/L开始,到1 mg/L达最大效果,呈剂量依赖性.而且,黄芪多糖能够抑制线粒体膜电位崩解保护线粒体形态,保护溶酶体膜.结论:这些结果证明了黄芪多糖在线粒体和溶酶体水平存在1个新的调节凋亡的途径.这个途径可以成为一些氧化应激以及溶酶体缺陷疾病的1个新的治疗靶点.

  3. 转KAP6.1-GFP-蜘蛛拖丝蛋白基因核心序列4S绵羊成纤维细胞株的筛选%Filtration of Transgenic Sheep Skin Fibroblasts with KAP6.1-GFP-polymerized Spider Dragline Silk Protein Gene(4S)

    Institute of Scientific and Technical Information of China (English)

    王春生; 原璐; 宁方勇; 吴治昊; 朴善花; 安铁洙

    2011-01-01

    [目的]通过体细胞核移植为获得皮肤特异表达蜘蛛拖丝蛋白的绵羊莫定基础.[方法]将pcDNA3.1和带有角蛋白结合蛋白启动子质粒pGM-T-KAP6.1分别用Bg1 Ⅱ和Hin d Ⅲ双酶切后连接,再与蜘蛛拖丝蛋白基因核心序列4S连接,最后通过酶切与pIRES2-EGFP质粒连接后构建真核表达载体pIRES2-EGFP-4S;将此载体线性化后,采用脂质体法转染绵羊皮肤成纤维细胞,通过G418筛选获得转基因阳性细胞.[结果]筛选得到转pIRES2-EGFP-4S的阳性细胞.对阳性细胞经体外培养后的检测显示:(1)细胞形态(长梭形)、细胞生长曲线(呈S形)、群体倍增时间(随培养时间增加逐渐缩短)和细胞接种率(细胞贴壁率及存活率在24 h内逐渐升高,并达到最高值125%)等均具有正常绵羊成纤维细胞的生物学特征;(2)阳性细胞经冷冻复苏后具有与新鲜阳性细胞相似的生物学特征;(3)PCR检测结果显示,pIRES2-EGFP-4S在阳性细胞的基因组中整合.[结论]获得具有在绵羊皮肤特异表达,且便于检测的转蜘蛛拖丝蛋白4S的绵羊成纤维细胞株.%[Objective] This study aims to establish transgenic sheep fibroblast cell line and lay a foundation for transgenic sheep with expression spider dragline silk protein gene in hair follicle by nuclear transplantation. [Method] pcDNA3.1 and pGM-T-KAP6.1(hair follicle-specific promoter) were digested by 5g/Ⅱand Hind Ⅲ, and linked each other. The recombinant plasmid was linked with spider dragline silk protein gene and then linked with pIRES2-EGFP by a series of molecular methods. The eukaryotic expression vector pIRES2-EGFP-4S was constructed. Sheep fibroblasts were transfected with the plasmid by cationic liposome method and G418 was used to filtrate them. After identified, transgenic cell line with spider dragline silk protein gene was established. [Result] The G418 positive cells were detected in vitro. The results showed that cellular morphology was similar

  4. 自体皮肤成纤维细胞与毛发角蛋白复合填充材料在医学美容中的应用%Application of complex filling material consisting of autologous skin fibroblast and hair keratin in medical cosmetology

    Institute of Scientific and Technical Information of China (English)

    赵娟; 李希宁; 李相军; 杨旻; 任立群

    2012-01-01

    目的 探讨自体皮肤成纤维细胞(Fibroblasts,Fbs)与毛发角蛋白(Hair keratin,HK)复合填充材料在面部除皱中的作用,为其在医学美容中的应用提供实验依据.方法 采用消化分离法对人Fbs进行原代培养,并按不同比例与HK颗粒进行复合培养,台盼蓝排斥法检测细胞的存活率,MTT法检测细胞的增殖活力,ELISA法检测培养液中Ⅰ型胶原蛋白(Collagen Ⅰ,Col Ⅰ)的含量.将志愿者耳后部Fbs与HK颗粒共培养后回注至面部皱纹真皮层内,观察除皱效果.结果 HK与Fbs的比例为1:2时,细胞的存活率、增殖活力及培养液中的Col Ⅰ含量均最高;志愿者术后4周,注射部位手感柔软,顺应性良好,无僵硬、挛缩等情况出现,皱纹的填充效果良好,外形改善明显.结论 自体Fbs与HK复合填充材料能明显促进面部皱纹的修复,本研究为其临床应用提供了实验依据.%Objective To investigate the role of complex filling material consisting of autologous skin fibroblast (Fbs) and hair keratin (HK) in removal of facial wrinkles and provide an experimental basis for application of the material in medical cosmetology. Methods Fbs isolated from volunteers were subjected to primary culture by digestion method, then to complex culture with HK particles at various ratios, and determined for survival rate by trypan blue exclusion method, for proliferation activity by MTT method, and for collagen I (Col I ) content in medium by ELJSA. The autologous Fbs extracted from opisthotic skins of 10 volunteers were co-cultured with HK particles and injected intradermally into the facial wrinkles to observe the wrinkle-removal effect. Results When the ratio of HK and Fbs was 1 : 2, the survival rate and proliferation activity of Fbs as well as the Col I content in medium reached peak value. Four weeks after injection, the skins at injection sites of volunteers were soft and of good compliance, while no stiffness or contracture appeared

  5. Skin Photoaging and the Role of Antioxidants in Its Prevention

    OpenAIRE

    Pandel, Ruža; Poljšak, Borut; Godic, Aleksandar; Dahmane, Raja

    2013-01-01

    Photoaging of the skin depends primarily on the degree of ultraviolet radiation (UVR) and on an amount of melanin in the skin (skin phototype). In addition to direct or indirect DNA damage, UVR activates cell surface receptors of keratinocytes and fibroblasts in the skin, which leads to a breakdown of collagen in the extracellular matrix and a shutdown of new collagen synthesis. It is hypothesized that dermal collagen breakdown is followed by imperfect repair that yields a deficit in the stru...

  6. 系统性硬皮病患者外周血单个核细胞对皮肤克隆成纤维细胞胶原代谢的影响%Effects of Peripheral Blood Mononuclear Cells on the Collagen Metabolism of Cloned Systemic Scleroderma Skin Fibroblasts

    Institute of Scientific and Technical Information of China (English)

    高地; 朱鹭冰; 李明

    2012-01-01

    目的:探讨系统性硬皮病(systemic scleroderma,SSc)患者外周血单个核细胞(peripheral blood mononuclear cells,PB-MCs)对具有不同胶原合成能力的皮肤克隆成纤维细胞的胶原代谢的影响.方法:采用实时荧光定量逆转录-聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)法检测系统性硬皮病患者和健康对照者PBMC培养上清液对具有不同胶原合成能力的皮肤克隆成纤维细胞中的Ⅰ型前胶原和基质金属蛋白酶-1(matrix metalloproteinase-1,MMP-1)的mR-NA水平的影响.结果:在体外培养的系统性硬皮病皮肤克隆成纤维细胞中加入10%患者PBMC培养上清液继续培养48 h仅使具有低胶原合成能力的SSc克隆成纤维细胞的Ⅰ型前胶原和MMP-1的mRNA水平上升[(0.70±0.29)比(0.07±0.08),P=0.001;(0.75±0.16)比(0.09±0.20),P<0.001];而加入10%健康对照PBMC培养上清液继续培养48h,仅使具有高胶原合成能力的SSc克隆成纤维细胞的Ⅰ型前胶原的mRNA水平下降[(-0.24±0.08)比(0.12±0.10),P=0.024],同时使具有高、低胶原合成能力的SSc克隆成纤维细胞的MMP-1 mRNA水平上升(P=0.003;P<0.001),且具有低胶原合成能力的克隆成纤维细胞MMP-1 mRNA水平上升幅度大[(1.27±0.23)比(0.56±0.08),P=0.000].结论:SSc具有不同胶原合成能力的皮肤克隆成纤维细胞的Ⅰ型前胶原和MMP-1的mRNA水平对PBMC的反应存在异质性.%Objective: To study the effects of peripheral hlood mononuclear cells (PBMCs) on the collagen metabolism of cloned systemic scleroderma (SSc) skin fibroblasts with heterogeneity in the synthesis of collagen. Methods:By means of realtime reverse transcription-polymerase chain reaction (RT-PCR),the effects of PBMC supernatants (from SSc patients and controls) on the expression of type I procollagen and matrix metalloproteinase-1 (MMP-1) on mRNA level in cloned SSc skin fibroblasts with heterogeneity in the synthesis of collagen were

  7. 胎肝间充质干细胞和皮肤成纤维细胞有限细胞系的建立及肝向分化研究%Establishment and hepatocyte differentiation of fetal liver mesenchymal stem cells and skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    朱争艳; 杜智; 骆莹; 黎娇; 王鹏; 刘彤; 高英堂

    2011-01-01

    Objective To establish a long-term culture system of fetal liver mesenchymal stem cells and skin fibroblasts, and to compare the phenotyping and the hepatocyte differentiation between two types of cells. Methods Fetus liver-derived mesenchymal stem cells were selected by adherence culture, and fibroblasts were isolated and cultured from skin tissue of the same individual. Immunocytochemistry (ICC)and flow cytometry (FCM) were used in detection of the expressions of CD34, CD90 and CD105, and chromosome analysis and soft agar colony formation test in study of biological characteristics in the two types of cells. The induced differentiation of the P3- 30 cell into hepatocyte was performed with hepatic culture medium (HCM) containing hepatocyte growth factor (HGF) , fibroblast growth factor 4 (FGF4) and oncostatin M(OSM). Albumin(ALB), CK18 and CK19 in the induced and non-induced cells were detected by ICC, glycogen stained by PAS, and the expressions of AFP and ALB mRNA by RT-PCR. Results CD90was highly expressed in fetal liver mesenchymal stem cells but barely detectable in skin fibroblasts. Culture in conditioned medium led to hepatocyte-like differentiation in approximately (5.1±0.2)% of fibroblasts versus ( 10.3±0.3)% of fetal liver mesenchymal stem cells (P<0.01). Both of the two types of cells could be cultured in vitro for about 50 passages that appeared karyotypically normal, non-oncogenic but not immortal.Conclusion Mesenchymal stem cells of adult tissues derived from any adult tissue may have a limited lifetime over a long-term culture in vitro, and some from certain adult tissues may show a stronger potential of differentiation to parenchyma cells of the origin.%通过体外长期培养胎肝间充值干细胞和皮肤成纤维细胞,建立两种细胞长期培养体系并比较此两种细胞的表型特征及向肝细胞分化的潜能。方法 从人胎肝中分离贴壁生长的间充质干细胞,同时取同一个体的皮肤组织,分离培

  8. Strong optical and UV intermediate-width emission lines in the quasar SDSS J232444.80-094600.3: dust-free and intermediate-density gas at the skin of dusty torus?

    Science.gov (United States)

    Li, Zhen-Zhen; Zhou, Hong-Yan; Hao, Lei; Wang, Shu-Fen; Ji, Tuo; Liu, Bo

    2016-09-01

    Emission lines from the broad emission line region (BELR) and the narrow emission line region (NELR) of active galactic nuclei (AGNs) have been extensively studied. However, emission lines are rarely detected between these two regions. We present a detailed analysis of quasar SDSS J232444.80-094600.3 (SDSS J2324-0946), which is remarkable for its strong intermediate-width emission lines (IELs) with FWHM ≈ 1800 km s-1. The IEL component is present in different emission lines, including the permitted lines Lyα λ1216, CIV λ1549, semiforbidden line [CIII] λ1909, and forbidden lines [OIII] λλ4959, 5007. With the aid of photo-ionization models, we found that the IELs are produced by gas with a hydrogen density of nH ˜ 106.2 ˜ 106.3 cm-3, a distance from the central ionizing source of R ˜ 35 - 50 pc, a covering factor of ˜ 6%, and a dust-to-gas ratio of ≤ 4% that of the SMC. We suggest that the strong IELs of this quasar are produced by nearly dust-free and intermediate-density gas located at the skin of the dusty torus. Such strong IELs, which serve as a useful diagnostic, can provide an avenue to study the properties of gas between the BELR and the NELR.

  9. Serotonin in human skin

    Institute of Scientific and Technical Information of China (English)

    Jianguo Huang; Qiying Gong; Guiming Li

    2005-01-01

    In this review the authors summarize data of a potential role for serotonin in human skin physiology and pathology. The uncovering of endogenous serotonin synthesis and its transformation to melatonin underlines a putative important role of this pathway in melanocyte physiology and pathology. Pathways of the biosynthesis and biodegradation of serotonin have been characterized in human beings and its major cellular populations. Moreover, receptors of serotonin are expressed on keratinocytes, melanocytes, and fibroblasts and these mediate phenotypic actions on cellular proliferation and differentiation. And the widespread expression of a cutaneous seorotoninergic system indicates considerable selectivity of action to facilitate intra-, auto-, or paracrine mechanisms that define and influence skin function in a highly compartmentalized manner. Melatonin, in turn, can also act as a hormone, neurotransmitter, cytokine, biological modifier and immunomodulator. Thus, Serotonin local synthesis and cellular localization could thus become of great importance in the diagnosis and management of cutaneous pathology.

  10. Periostin induces fibroblast proliferation and myofibroblast persistence in hypertrophic scarring.

    Science.gov (United States)

    Crawford, Justin; Nygard, Karen; Gan, Bing Siang; O'Gorman, David Brian

    2015-02-01

    Hypertrophic scarring is characterized by the excessive development and persistence of myofibroblasts. These cells contract the surrounding extracellular matrix resulting in the increased tissue density characteristic of scar tissue. Periostin is a matricellular protein that is abnormally abundant in fibrotic dermis, however, its roles in hypertrophic scarring are largely unknown. In this report, we assessed the ability of matrix-associated periostin to promote the proliferation and myofibroblast differentiation of dermal fibroblasts isolated from the dermis of hypertrophic scars or healthy skin. Supplementation of a thin type-I collagen cell culture substrate with recombinant periostin induced a significant increase in the proliferation of hypertrophic scar fibroblasts but not normal dermal fibroblasts. Periostin induced significant increases in supermature focal adhesion formation, α smooth muscle actin levels and collagen contraction in fibroblasts cultured from hypertrophic scars under conditions of increased matrix tension in three-dimensional type-I collagen lattices. Inhibition of Rho-associated protein kinase activity significantly attenuated the effects of matrix-associated periostin on hypertrophic scar fibroblasts and myofibroblasts. Depletion of endogenous periostin expression in hypertrophic scar myofibroblasts resulted in a sustained decrease in α smooth muscle actin levels under conditions of reducing matrix tension, while matrix-associated periostin levels caused the cells to retain high levels of a smooth muscle actin under these conditions. These findings indicate that periostin promotes Rho-associated protein kinase-dependent proliferation and myofibroblast persistence of hypertrophic scar fibroblasts and implicate periostin as a potential therapeutic target to enhance the resolution of scars.

  11. Increased fibroblast functionality on CNN2-loaded titania nanotubes.

    Science.gov (United States)

    Wei, Hongbo; Wu, Shuyi; Feng, Zhihong; Zhou, Wei; Dong, Yan; Wu, Guofeng; Bai, Shizhu; Zhao, Yimin

    2012-01-01

    Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant-skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor) fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.

  12. 张力刺激皮肤成纤维细胞向肌成纤维细胞分化的实验研究%Myofibroblasts Differentiation of Skin Fibroblasts Induced by Mechanical Loading Based on 3D Culture in Vitro

    Institute of Scientific and Technical Information of China (English)

    傅士博; 温从吉; 王琛; 王丹茹

    2014-01-01

    目的:探索机械张力刺激诱导基于三维培养的皮肤成纤维细胞向肌成纤维细胞分化的可行性。方法将正常人皮肤成纤维细胞三维培养于Ⅰ型鼠尾胶原支架中,通过机械张力装置进行拉伸实验(牵拉组,n=9),并设立对照(非牵拉组,n=9),绘制细胞生长曲线,免疫荧光染色和实时定量PCR检测成纤维细胞的分化程度。结果三维培养环境下细胞增殖缓慢,牵拉组有促进细胞增殖的趋势,但与非牵拉组相比,统计学差异不显著;免疫荧光染色显示,牵拉组成纤维细胞α-SMA和fibronectin染色亮度明显高于非牵拉组;实时定量PCR检测显示,牵拉组α-SMA、纤连蛋白和Ⅰ、Ⅲ型胶原mRNA表达高于非牵拉组。结论机械张力可成功诱导基于三维培养的皮肤成纤维细胞向肌成纤维细胞分化。%Objective To explore the feasibility of inducing myofibroblasts differentiation of skin fibroblasts by mechanical loading based on three dimensional culture in vitro. Methods Human dermal fibroblasts were isolated and three dimensionally cultured in type I rat tail collagen scaffold. Then mechanical loading were added by stretching the gel (stretch group, n=9), and control group was set up (non-stretch group, n=9). The situation of myofibroblasts differentiation were tested by drawing cell growth curve, immunofluorescent staining and Real-Time PCR. Results Cell proliferation was slowed down under mechanical loading environment. The cell proliferation was promoted in stretch group, but there was no significant difference comparing with non-stretch group. Dyeing brightness of α-SMA and fibronectin were significantly higher in stretch group showed by immunofluorescent staining. Higher mRNA expression of α-SMA, fibronectin, type I and III collagen genes were observed in stretch group by real time PCR. Conclusion The model of mechanical loading inducing myofibroblasts differentiation of skin

  13. Skin Infections

    Science.gov (United States)

    ... Old Feeding Your 8- to 12-Month-Old Feeding Your 1- to 2-Year-Old First Aid: Skin ... (bacterial infection of the deeper layers of the skin and tissues beneath) are typical childhood skin infections. The usual bacterial culprits in skin ...

  14. Skin Biomes.

    Science.gov (United States)

    Fyhrquist, N; Salava, A; Auvinen, P; Lauerma, A

    2016-05-01

    The cutaneous microbiome has been investigated broadly in recent years and some traditional perspectives are beginning to change. A diverse microbiome exists on human skin and has a potential to influence pathogenic microbes and modulate the course of skin disorders, e.g. atopic dermatitis. In addition to the known dysfunctions in barrier function of the skin and immunologic disturbances, evidence is rising that frequent skin disorders, e.g. atopic dermatitis, might be connected to a dysbiosis of the microbial community and changes in the skin microbiome. As a future perspective, examining the skin microbiome could be seen as a potential new diagnostic and therapeutic target in inflammatory skin disorders.

  15. GHK Peptide as a Natural Modulator of Multiple Cellular Pathways in Skin Regeneration

    Directory of Open Access Journals (Sweden)

    Loren Pickart

    2015-01-01

    Full Text Available GHK (glycyl-L-histidyl-L-lysine is present in human plasma, saliva, and urine but declines with age. It is proposed that GHK functions as a complex with copper 2+ which accelerates wound healing and skin repair. GHK stimulates both synthesis and breakdown of collagen and glycosaminoglycans and modulates the activity of both metalloproteinases and their inhibitors. It stimulates collagen, dermatan sulfate, chondroitin sulfate, and the small proteoglycan, decorin. It also restores replicative vitality to fibroblasts after radiation therapy. The molecule attracts immune and endothelial cells to the site of an injury. It accelerates wound-healing of the skin, hair follicles, gastrointestinal tract, boney tissue, and foot pads of dogs. It also induces systemic wound healing in rats, mice, and pigs. In cosmetic products, it has been found to tighten loose skin and improve elasticity, skin density, and firmness, reduce fine lines and wrinkles, reduce photodamage, and hyperpigmentation, and increase keratinocyte proliferation. GHK has been proposed as a therapeutic agent for skin inflammation, chronic obstructive pulmonary disease, and metastatic colon cancer. It is capable of up- and downregulating at least 4,000 human genes, essentially resetting DNA to a healthier state. The present review revisits GHK’s role in skin regeneration in the light of recent discoveries.

  16. Immunohistochemistry and in situ hybridization in the study of human skin melanocytes.

    Science.gov (United States)

    Passeron, Thierry; Coelho, Sergio G; Miyamura, Yoshinori; Takahashi, Kaoruko; Hearing, Vincent J

    2007-03-01

    Although keratinocytes are the most numerous type of cell in the skin, melanocytes are also key players as they produce and distribute melanin that protects the skin from ultraviolet (UV) radiation. In vitro experiments on melanocytic cell lines are useful to study melanogenesis and their progression towards melanoma. However, interactions of melanocytes with keratinocytes and with other types of cells in the skin, such as fibroblasts and Langerhans cells, are also crucial. We describe two techniques, immunohistochemistry (IHC) and tissue in situ hybridization (TISH), that can be used to identify and study melanocytes in the skin and their responses to UV or other stimuli in situ. We describe a practical method to localize melanocytic antigens on formalin-fixed, paraffin-embedded tissue sections and in frozen sections using indirect immunofluorescence with conjugated secondary antibodies. In addition, we detail the use of TISH and its combination with IHC to study mRNA levels of genes expressed in the skin at cellular resolution. This methodology, along with relevant tips and troubleshooting items, are important tools to identify and study melanocytes in the skin.

  17. Activation of the innate immune response against DENV in normal non-transformed human fibroblasts.

    Directory of Open Access Journals (Sweden)

    José Bustos-Arriaga

    2011-12-01

    Full Text Available BACKGROUND: When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times ("probing" before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells. METHODOLOGY/PRINCIPAL FINDINGS: Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5 and β defensin 2 (HβD2. In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN regulatory factor 3 (IRF3, but not interferon regulatory factor 7 (IRF7, when compared with mock-infected fibroblasts. CONCLUSIONS/SIGNIFICANCE: In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and

  18. Deguelin‐induced blockade of PI3K/protein kinase B/MAP kinase signaling in zebrafish and breast cancer cell lines is mediated by down‐regulation of fibroblast growth factor receptor 4 activity

    OpenAIRE

    Wu, Wei; Hai, Yang; Chen, Lu; Liu, Rui‐Jin; Han, Yu‐Xiang; Li, Wen‐Hao; Li, Song; Lin, Shuo; Wu, Xin‐Rong

    2016-01-01

    Abstract Deguelin, a natural component derived from leguminous plants, has been used as pesticide in some regions. Accumulating evidence show that deguelin has promising chemopreventive and therapeutic activities against cancer cells. This study shows that low concentrations of deguelin can lead to significant delay in zebrafish embryonic development through growth inhibition and induction of apoptosis. Furthermore, we identified fibroblast growth factor receptor 4 (FGFR4) as the putative tar...

  19. Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

    Directory of Open Access Journals (Sweden)

    Gennaro Altamura

    2013-01-01

    Full Text Available Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1 and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm.

  20. Photoprotective potential of strawberry (Fragaria × ananassa) extract against UV-A irradiation damage on human fibroblasts.

    Science.gov (United States)

    Giampieri, Francesca; Alvarez-Suarez, Josè M; Tulipani, Sara; Gonzàles-Paramàs, Ana M; Santos-Buelga, Celestino; Bompadre, Stefano; Quiles, José L; Mezzetti, Bruno; Battino, Maurizio

    2012-03-07

    Exposure to UV-A radiation is known to induce discrete lesions in DNA and the generation of free radicals that lead to a wide array of skin diseases. Strawberry (Fragaria × ananassa) contains several polyphenols with strong antioxidant and anti-inflammatory activities. Because the major representative components of strawberry are anthocyanins, these may significantly contribute to its properties. To test this hypothesis, methanolic extracts from the Sveva cultivar were analyzed for anthocyanin content and for their ability to protect human dermal fibroblasts against UV-A radiation, as assayed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenytetrazolium bromide and Comet assays. Five anthocyanin pigments were identified using high-performance liquid chromatography-diode array detection-electrospray ionization/mass spectrometry. Moreover, the strawberry extract showed a photoprotective activity in fibroblasts exposed to UV-A radiation, increasing cellular viability, and diminishing DNA damage, as compared to control cells. Overall, our data show that strawberry contains compounds that confer photoprotective activity in human cell lines and may protect skin against the adverse effects of UV-A radiation.

  1. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Directory of Open Access Journals (Sweden)

    Thomas Zuliani

    Full Text Available Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  2. Basal Cell Carcinoma in Gorlin's Patients: a Matter of Fibroblasts-Led Protumoral Microenvironment?

    Directory of Open Access Journals (Sweden)

    Yannick Gache

    Full Text Available Basal cell carcinoma (BCC is the commonest tumor in human. About 70% sporadic BCCs bear somatic mutations in the PATCHED1 tumor suppressor gene which encodes the receptor for the Sonic Hedgehog morphogen (SHH. PATCHED1 germinal mutations are associated with the dominant Nevoid Basal Cell Carcinoma Syndrome (NBCCS, a major hallmark of which is a high susceptibility to BCCs. Although the vast majority of sporadic BCCs arises exclusively in sun exposed skin areas, 40 to 50% BCCs from NBCCS patients develop in non photo-exposed skin. Since overwhelming evidences indicate that microenvironment may both be modified by- and influence the- epithelial tumor, we hypothesized that NBCCS fibroblasts could contribute to BCCs in NBCCS patients, notably those developing in non photo-exposed skin areas. The functional impact of NBCCS fibroblasts was then assessed in organotypic skin cultures with control keratinocytes. Onset of epidermal differentiation was delayed in the presence of primary NBCCS fibroblasts. Unexpectedly, keratinocyte proliferation was severely reduced and showed high levels of nuclear P53 in both organotypic skin cultures and in fibroblast-led conditioning experiments. However, in spite of increased levels of senescence associated β-galactosidase activity in keratinocytes cultured in the presence of medium conditioned by NBCCS fibroblasts, we failed to observe activation of P16 and P21 and then of bona fide features of senescence. Constitutive extinction of P53 in WT keratinocytes resulted in an invasive phenotype in the presence of NBCCS fibroblasts. Finally, we found that expression of SHH was limited to fibroblasts but was dependent on the presence of keratinocytes. Inhibition of SHH binding resulted in improved epidermal morphogenesis. Altogether, these data suggest that the repertoire of diffusible factors (including SHH expressed by primary NBCCS fibroblasts generate a stress affecting keratinocytes behavior and epidermal homeostasis

  3. Fetal fibroblasts and keratinocytes with immunosuppressive properties for allogeneic cell-based wound therapy.

    Science.gov (United States)

    Zuliani, Thomas; Saiagh, Soraya; Knol, Anne-Chantal; Esbelin, Julie; Dréno, Brigitte

    2013-01-01

    Fetal skin heals rapidly without scar formation early in gestation, conferring to fetal skin cells a high and unique potential for tissue regeneration and scar management. In this study, we investigated the possibility of using fetal fibroblasts and keratinocytes to stimulate wound repair and regeneration for further allogeneic cell-based therapy development. From a single fetal skin sample, two clinical batches of keratinocytes and fibroblasts were manufactured and characterized. Tolerogenic properties of the fetal cells were investigated by allogeneic PBMC proliferation tests. In addition, the potential advantage of fibroblasts/keratinocytes co-application for wound healing stimulation has been examined in co-culture experiments with in vitro scratch assays and a multiplex cytokines array system. Based on keratin 14 and prolyl-4-hydroxylase expression analyses, purity of both clinical batches was found to be above 98% and neither melanocytes nor Langerhans cells could be detected. Both cell types demonstrated strong immunosuppressive properties as shown by the dramatic decrease in allogeneic PBMC proliferation when co-cultured with fibroblasts and/or keratinocytes. We further showed that the indoleamine 2,3 dioxygenase (IDO) activity is required for the immunoregulatory activity of fetal skin cells. Co-cultures experiments have also revealed that fibroblasts-keratinocytes interactions strongly enhanced fetal cells secretion of HGF, GM-CSF, IL-8 and to a lesser extent VEGF-A. Accordingly, in the in vitro scratch assays the fetal fibroblasts and keratinocytes co-culture accelerated the scratch closure compared to fibroblast or keratinocyte mono-cultures. In conclusion, our data suggest that the combination of fetal keratinocytes and fibroblasts could be of particular interest for the development of a new allogeneic skin substitute with immunomodulatory activity, acting as a reservoir for wound healing growth factors.

  4. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

    Science.gov (United States)

    Härle-Bachor, C; Boukamp, P

    1996-06-25

    Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

  5. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    Science.gov (United States)

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

  6. Skin Cancer

    Science.gov (United States)

    ... are round and lie directly under squamous cells. Melanocytes are specialized skin cells that produce pigment called melanin. The melanin pigment produced by melanocytes gives skin its color. It also protects the ...

  7. Skin Cancer

    Science.gov (United States)

    ... the head, face, neck, hands, and arms. Another type of skin cancer, melanoma, is more dangerous but less common. Anyone ... cancer is found early. If not treated, some types of skin cancer cells can spread to other tissues and organs. ...

  8. Sagging Skin

    Science.gov (United States)

    ... Dermatologic Surgery Expertise for the life of your skin (TM) Public Resources Dermatologic Surgery Conditions Treatments and ... learn-more"> 3 million Americans are diagnosed with skin cancer every year Any suspicious new growths or ...

  9. Skin tears.

    Science.gov (United States)

    Baranoski, S

    2001-08-01

    Skin tears are a serious, painful problem for older patients. Find out how your staff can recognize patients at risk, what they can do to prevent skin tears, and how to manage them effectively if they occur.

  10. Skin turgor

    Science.gov (United States)

    ... arm or abdomen is checked. The skin is held for a few seconds then released. Skin with ... University of Washington, Seattle, WA. Also reviewed by David Zieve, MD, MHA, Isla Ogilvie, PhD, and the ...

  11. Periostin in Skin Tissue Skin-Related Diseases

    Directory of Open Access Journals (Sweden)

    Yukie Yamaguchi

    2014-01-01

    Recently, periostin—a matricellular protein—has been highlighted for its pivotal functions in the skin. Analysis of periostin null mice has revealed that periostin contributes to collagen fibrillogenesis, collagen cross-linking, and the formation of ECM meshwork via interactions with other ECM components. Periostin expression is enhanced by mechanical stress or skin injury; this is indicative of the physiologically protective functions of periostin, which promotes wound repair by acting on keratinocytes and fibroblasts. Along with its physiological functions, periostin plays pathogenic roles in skin fibrosis and chronic allergic inflammation. In systemic sclerosis (SSc patients, periostin levels reflect the severity of skin fibrosis. Periostin null mice have shown reduced skin fibrosis in a bleomycin-induced SSc mouse model, indicating a key role of periostin in fibrosis. Moreover, in atopic dermatitis (AD, attenuated AD phenotype has been observed in periostin null mice in a house dust mite extract-induced AD mouse model. Th2 cytokine-induced periostin acts on keratinocytes to produce inflammatory cytokines that further enhance the Th2 response, thereby sustaining and amplifying chronic allergic inflammation. Thus, periostin is deeply involved in the pathogenesis of AD and other inflammation-related disorders affecting the skin. Understanding the dynamic actions of periostin would be key to dissecting pathogenesis of skin-related diseases and to developing novel therapeutic strategies.

  12. Peripheral intravenous line (image)

    Science.gov (United States)

    A peripheral intravenous line is a small, short plastic catheter that is placed through the skin into a vein, ... or foot, but occasionally in the head. A peripheral intravenous line is used to give fluids and ...

  13. Peripheral arterial line (image)

    Science.gov (United States)

    A peripheral arterial line is a small, short plastic catheter placed through the skin into an artery of the arm or leg. The purpose of a peripheral arterial line is to allow continuous monitoring of ...

  14. Adhesion, growth, and matrix production by fibroblasts on laminin substrates

    DEFF Research Database (Denmark)

    Couchman, J R; Höök, M; Rees, D A;

    1983-01-01

    Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin and lami......Human embryonic skin fibroblasts have been shown to attach and spread on laminin substrates in the absence of protein synthesis and presence of fibronectin-depleted serum and anti-fibronectin antibodies. Rates of attachment and the type of spreading are virtually identical on fibronectin...... and laminin-coated substrates with the development of microfilament bundles and focal adhesions. Antibodies to laminin, but not fibronectin, will prevent or reverse fibroblast adhesion to laminin, whereas antibodies to fibronectin but not laminin will give similar results on fibronectin-coated substrates....... These and other results indicate that fibroblasts possess distinct receptors for laminin and fibronectin which on contact with suitable substrates promote adhesion through interaction with common intermediates. This type of adhesion is compatible with subsequent growth and extracellular matrix production....

  15. Differential expression of wound fibrotic factors between facial and trunk dermal fibroblasts.

    Science.gov (United States)

    Kurita, Masakazu; Okazaki, Mutsumi; Kaminishi-Tanikawa, Akiko; Niikura, Mamoru; Takushima, Akihiko; Harii, Kiyonori

    2012-01-01

    Clinically, wounds on the face tend to heal with less scarring than those on the trunk, but the causes of this difference have not been clarified. Fibroblasts obtained from different parts of the body are known to show different properties. To investigate whether the characteristic properties of facial and trunk wound healing are caused by differences in local fibroblasts, we comparatively analyzed the functional properties of superficial and deep dermal fibroblasts obtained from the facial and trunk skin of seven individuals, with an emphasis on tendency for fibrosis. Proliferation kinetics and mRNA and protein expression of 11 fibrosis-associated factors were investigated. The proliferation kinetics of facial and trunk fibroblasts were identical, but the expression and production levels of profibrotic factors, such as extracellular matrix, transforming growth factor-β1, and connective tissue growth factor mRNA, were lower in facial fibroblasts when compared with trunk fibroblasts, while the expression of antifibrotic factors, such as collagenase, basic fibroblast growth factor, and hepatocyte growth factor, showed no clear trends. The differences in functional properties of facial and trunk dermal fibroblasts were consistent with the clinical tendencies of healing of facial and trunk wounds. Thus, the differences between facial and trunk scarring are at least partly related to the intrinsic nature of the local dermal fibroblasts.

  16. Skin Aging

    Science.gov (United States)

    Your skin changes as you age. You might notice wrinkles, age spots and dryness. Your skin also becomes thinner and loses fat, making it ... heal, too. Sunlight is a major cause of skin aging. You can protect yourself by staying out ...

  17. Age-related skin changes

    Directory of Open Access Journals (Sweden)

    Božanić Snežana

    2012-01-01

    Full Text Available Age-related skin changes can be induced by chronological ageing, manifested in subcutaneous fat reduction, and photo-ageing eliciting increased elastotic substance in the upper dermis, destruction of its fibrilar structure, augmented intercellular substance and moderate inflammatory infiltrate. Forty-five biopsy skin samples of the sun-exposed and sun-protected skin were analyzed. The patients were both males and females, aged from 17 to 81 years. The thickness of the epidermal layers and the number of cellular living layers is greater in younger skin. The amount of keratohyaline granules is enlarged in older skin. Dermoepidermal junction is flattened and the presence of elastotic material in the dermis is pronounced with age. The amount of inflammatory infiltrate is increased, the fibrous trabeculae are thickened in older skin and the atrophy of the hypodermis is observed. Chronological ageing alters the fibroblasts metabolism by reducing their life span, capacity to divide and produce collagen. During ageing, the enlargement of collagen fibrils diminishes the skin elasticity.

  18. Effect of mini Ad-ATP7B-GFP on the copper metabolism of skin fibroblasts of Wilson′s disease patients%重组微小腺病毒载体对 Wilson 病患者皮肤成纤维细胞铜代谢的影响

    Institute of Scientific and Technical Information of China (English)

    刘磊磊; 汤其强; 朱迎春

    2016-01-01

    Objective To explore the effect of miniAd-ATP7B-GFP on the copper metabolism of skin fibroblasts of Wilson′s disease ( WD ) patients under high concentration copper medium.Methods Firstly, mini-adenovirus carrier containing human ATP7B gene was built and the mutations of 8 WD patients were detected.Fibroblasts from primary culture of skin of WD patients and normal human were cultivated 72 h in basic medium and medium with the copper concentration of C1(22.3μmol/L), C2(89.2μmol/L), C3 (156.1 μmol/L), C4 (245.3 μmol/L).Then the concentration of copper and protein was detected and copper/protein ratio was calculated.Secondly, miniAd-GFP ( miniAd-GFP group) and miniAd-ATP7B-GFP ( miniAd-ATP7B-GFP group ) were added into WD patients skin fibroblasts respectively, Wilson non-transfection group and normal group were set up as control, and C4 medium was used to culture the cells of four groups for 72 h and 96 h.Then the concentration of copper and protein was detected and copper/protein ratio was calculated.Results Five kinds of mutations were detected from 8 WD patients.The copper/protein ratio of WD patients and normal human in basic medium and the C1 -C3 groups had no statistically significant difference, but in C4 group (WD (1 871.6 ±209.2) ng/mg, normal group (1 267.2 ±188.3) ng/mg) the difference was statistically significant (t=6.075, P<0.01).C3((816.3 ±113.9) ng/mg) and C4 groups had statistically significant difference compared with the basic medium group ( ( 159.2 ± 38.6) ng/mg;WD:χ2 =31.493, normal group:χ2 =30.708, both P<0.01).The copper/protein ratio of 96 h group was higher than 72 h group.Compared with WD non-transfection (96 h:(2 731.2 ±188.7) ng/mg,72 h:(1 901.7 ±219.5) ng/mg) and normal groups, miniAd-ATP7B-GFP group had statistically significant difference both in 96 h ( ( 2 071.0 ±171.8 ) ng/mg ) and 72 h groups ( ( 1 495.5 ±161.4 ) ng/mg;72 h:F=20.130, 96 h: F=51.496,P<0.01).Conclusion MiniAd-ATP7B-GFP has partial improvement on

  19. Morfometria de fibroblastos e fibrócitos durante o processo cicatricial na pele de coelhos da raça Nova Zelândia Branco tratados com calêndula Morphometry of fibroblasts and fibrocytes during wound healing in the skin of rabbits of the New Zeland White breed treated with marigold

    Directory of Open Access Journals (Sweden)

    Leonardo de Oliveira Pagnano

    2008-09-01

    Full Text Available O objetivo deste estudo foi avaliar a capacidade cicatrizante da calêndula (Calendula officinalis L. sobre feridas cutâneas experimentais, em 15 coelhos, distribuídos em três grupos denominados: excipiente, calêndula e controle. Cada animal foi submetido à uma incisão cirúrgica de 6cm de comprimento, lateral à coluna vertebral e suturada no padrão U. Os produtos avaliados foram colocados sobre as incisões durante sete dias na quantidade de 0,1ml (loção cremosa não-iônica - grupo excipiente; tintura de calêndula a 5% - grupo calêndula e nos animais do grupo controle não se utilizou nenhum produto. A biópsia de pele foi realizada no 1°, 3°, 5° e 7° dia após a incisão cirúrgica para avaliação morfométrica do processo cicatricial, analisando-se o número de fibroblastos e fibrócitos. A morfometria foi realizada por meio de microscópio óptico adaptado a um sistema computadorizado de análise de imagens. De acordo com os resultados, a calêndula propiciou obtenção dos maiores valores médios das células envolvidas no processo cicatricial, os fibroblastos, deduzindo que a mesma, inferiu uma resposta mais satisfatória na cicatrização em relação aos demais tratamentos.The aim of this study was to evaluate the scarring capability of marigold (Calendula officinalis L. on experimental skin wounds in 15 rabbits, distributed in three groups: excipient, marigold and control. Each animal was subjected to a surgical incision measuring 6cm in length, laterally to the spinal column and sutured in U-shape. Products evaluated were placed on the incisions for 7 days, at a rate of 0.1ml (nonionic creamy lotion - excipient group; 5% marigold extract and no treatment was provided to control animals. Skin biopsy was performed on 1, 3, 5, and 7 days after wounding, for morphometric and cicatricial process evaluations. The morphometry was performed with an optical microscope adapted to a computadorized picture analysis system. The

  20. Increased fibroblast functionality on CNN2-loaded titania nanotubes

    Directory of Open Access Journals (Sweden)

    Wei HB

    2012-02-01

    Full Text Available Hongbo Wei*, Shuyi Wu*, Zhihong Feng, Wei Zhou, Yan Dong, Guofeng Wu, Shizhu Bai, Yimin Zhao Department of Prosthodontics, School of Stomatology, Fourth Military Medical University, Xi'an, People's Republic of China *These authors contributed equally to this workAbstract: Infection and epithelial downgrowth are major problems associated with maxillofacial percutaneous implants. These complications are mainly due to the improper closure of the implant–skin interface. Therefore, designing a percutaneous implant that better promotes the formation of a stable soft tissue biologic seal around percutaneous sites is highly desirable. Additionally, the fibroblast has been proven to play an important role in the formation of biologic seals. In this study, titania nanotubes were filled with 11.2 kDa C-terminal CCN2 (connective tissue growth factor fragment, which could exert full CCN2 activity to increase the biological functionality of fibroblasts. This drug delivery system was fabricated on a titanium implant surface. CCN2 was loaded into anodized titania nanotubes using a simplified lyophilization method and the loading efficiency was approximately 80%. Then, the release kinetics of CCN2 from these nanotubes was investigated. Furthermore, the influence of CCN2-loaded titania nanotubes on fibroblast functionality was examined. The results revealed increased fibroblast adhesion at 0.25, 0.5, 1, 2, 4, and 24 hours, increased fibroblast viability over the course of 5 days, as well as enhanced actin cytoskeleton organization on CCN2-loaded titania nanotubes surfaces compared to uncoated, unmodified counterparts. Therefore, the results from this in vitro study demonstrate that CCN2-loaded titania nanotubes have the ability to increase fibroblast functionality and should be further studied as a method of promoting the formation of a stable soft tissue biologic seal around percutaneous sites.Keywords: anodization, titania nanotubes, adhesion, connective

  1. Primary fibroblasts of NDUFS4(-/-) mice display increased ROS levels and aberrant mitochondrial morphology.

    Science.gov (United States)

    Valsecchi, Federica; Grefte, Sander; Roestenberg, Peggy; Joosten-Wagenaars, Jori; Smeitink, Jan A M; Willems, Peter H G M; Koopman, Werner J H

    2013-09-01

    The human NDUFS4 gene encodes an accessory subunit of the first mitochondrial oxidative phosphorylation complex (CI) and, when mutated, is associated with progressive neurological disorders. Here we analyzed primary muscle and skin fibroblasts from NDUFS4(-/-) mice with respect to reactive oxygen species (ROS) levels and mitochondrial morphology. NDUFS4(-/-) fibroblasts displayed an inactive CI subcomplex on native gels but proliferated normally and showed no obvious signs of apoptosis. Oxidation of the ROS sensor hydroethidium was increased and mitochondria were less branched and/or shorter in NDUFS4(-/-) fibroblasts. We discuss the relevance of these findings with respect to previous results and therapy development.

  2. Gene targeting in adult rhesus macaque fibroblasts

    Directory of Open Access Journals (Sweden)

    Wolf Don P

    2008-03-01

    Full Text Available Abstract Background Gene targeting in nonhuman primates has the potential to produce critical animal models for translational studies related to human diseases. Successful gene targeting in fibroblasts followed by somatic cell nuclear transfer (SCNT has been achieved in several species of large mammals but not yet in primates. Our goal was to establish the protocols necessary to achieve gene targeting in primary culture of adult rhesus macaque fibroblasts as a first step in creating nonhuman primate models of genetic disease using nuclear transfer technology. Results A primary culture of adult male fibroblasts was transfected with hTERT to overcome senescence and allow long term in vitro manipulations. Successful gene targeting of the HPRT locus in rhesus macaques was achieved by electroporating S-phase synchronized cells with a construct containing a SV40 enhancer. Conclusion The cell lines reported here could be used for the production of null mutant rhesus macaque models of human genetic disease using SCNT technology. In addition, given the close evolutionary relationship and biological similarity between rhesus macaques and humans, the protocols described here may prove useful in the genetic engineering of human somatic cells.

  3. STUDY ON FUNCTION OF FOCAL ADHENSIVE KINASE AND INTEGRIN α1 IN HYPERTROPHIC SCAR FIBROBLASTS

    Institute of Scientific and Technical Information of China (English)

    FU Min-gang; PING Ping; FAN Zhi-hong

    2008-01-01

    Objective To study the function of focal adhesion kinase (FAK) in the formation of hyper-trophic scar and its interrelationship with integrin α1. Methods Original fibroblasts from human hypertrophic scar and human normal dermis were cultured, and immanocytochemistry was applied to detect localization of expres-sion of FAK and integrin α1 in hypertrophic scar and human normal skin fibroblasts. The expression of integrin α1 was detected before and after FAK antibody blocking hypertrophic scar fibroblasts (HSFB) 48 h later. Meanwhile the collagen synthesis was evaluated by [3H] -proline incorporation and HSFB cell proliferation was measured by MTT method. Results The expression of FAK and integrin α1 of hypertrophic scar fibroblasts was higher than that of the normal skin fibroblasts significantly (P <0. 01). The expression of integrinct, was reduced after FAK be-ing blocked (P<0.01). Meanwhile the collagen synthesis of human scar-derived fibroblasts by [3H] -proline incor-poration was depressed respectively (P<0.01). The cell proliferation was inhibited by using 1:100 and 1:200 FAK antibody with MTT method (P<0.01). Conclusion FAK is the key point of signal transmission pathway medi-ated by integrin α1, which regulates protein synthesis of integrin α1, it may play an important role in the prolifera-tion and constriction of hypertrophic scar. FAK antibody can inhibit the collagen synthesis and cell proliferation of hypertrophic scar fibroblasts.

  4. Heparin fragments modulate the collagen phenotype of fibroblasts from radiation-induced subcutaneous fibrosis

    Energy Technology Data Exchange (ETDEWEB)

    el Nabout, R.; Martin, M.; Remy, J.; Robert, L.; Lafuma, C. (Laboratoire de Biochimie de Tissu Conjonctif, Creteil (France))

    1989-10-01

    Acute local gamma irradiation of porcine skin induces, as in human skin, an extensive and mutilating sclerosis characterized by continuous expansion of the fibrosis invading the adjacent muscle and by accumulation of the macromolecular components of the extracellular matrix. Collagen synthesis, content, and types were studied in the presence of heparin fragments (100 micrograms/10(6) cells) in the culture medium, by measuring the incorporation of the radiolabeled precursor (3H)proline into confluent primary cultures of porcine fibroblasts obtained from normal and irradiated fibrotic dermis. Enhancement in collagen biosynthesis and deposition and preferential increase in collagen type III synthesis were observed in fibrotic fibroblast cultures when compared to those in normal dermis fibroblasts. The total collagen synthesis and the rate of collagen hydroxylation appear unmodified by heparin fragments both in normal and in fibrotic fibroblast cultures. But heparin fragments induce a 10- and 2-fold decrease, respectively, in collagen type III and type V syntheses by fibrosis fibroblasts. As only minor effects upon collagen type III and V are observed in cultures of normal dermis fibroblasts, these results highly suggest that heparin fragments are capable of specifically modulating the collagen phenotype of fibroblasts derived from radiation-induced dermis fibrosis and thus are able to regulate the fibrotic process.

  5. bFGF、FGF受体I在HEp-2喉癌和MGE803胃癌细胞株中表达的检测%DETECTION OF EXPRESSIONS OF BASIC FIBROBLAST GROWTH FACTOR(bFGF), and FIBROBLAST GROWTH FACTOR RECEPTER-1(FGFR-1) IN HEp-2 LARYGO- TUMOR AND MGE803 GASTRO-TUMOR CELL LINE

    Institute of Scientific and Technical Information of China (English)

    张颖; 吕华; 沐桂藩

    2002-01-01

    目的通过对碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF),成纤维细胞生长因子受体-1(fibroblast growth factor receptor-1, FGFR-1)的检测,揭示两者在体内外的表达以及与肿瘤形成的关系. 方法采用免疫组织化学的检测方法,检测了体外培养的HEP-2喉癌细胞和MGE803胃癌细胞以及由两者在裸鼠皮下形成的实体瘤组织中bFGF和FGFR-1的表达情况. 结果在体外培养的这两种肿瘤细胞中bFGF的阳性染色存在于整个细胞.FGFR-1的染色主要存在于细胞核.在两种肿瘤细胞形成的实体瘤组织中bFGF和FGFR-1阳性染色细胞主要是围绕于坏死区的肿瘤细胞. 结论此两种肿瘤细胞在体内外均能表达bFGF和FGFR-1.bFGF与其高亲和力受体FGFR-1结合后进入细胞核,并在细胞核内发挥其生物功能,参与了肿瘤组织的形成和发展.

  6. Influence of mechanical stimulation on human dermal fibroblasts derived from different body sites.

    Science.gov (United States)

    Kuang, Ruixia; Wang, Zhiguo; Xu, Quanchen; Liu, Su; Zhang, Weidong

    2015-01-01

    Mechanical stimulation is highly associated with pathogenesis of human hypertrophic scar. Although much work has focused on the influence of mechanical stress on fibroblast populations from various tissues and organs in the human body, their effects on cultured dermal fibroblasts by the area of the body have not been as well studied. In this study, cultures of skin fibroblasts from two different body sites were subjected to cyclic mechanical stimulation with a 10% stretching amplitude at a frequency of 0.1 Hz for 24, 36 and 48 hours, respectively, and thereafter harvested for experimental assays. Fibroblasts from scapular upper back skin, subjected to mechanical loads for 36 and 48 hours, respectively, were observed to proliferate at a higher rate and reach confluent more rapidly during in vitro culturing, had higher expression levels of mRNA and protein production of integrin β1, p130Cas and TGF β1 versus those from medial side of upper arm. These data indicate that skin fibroblasts, with regard to originated body sites studied in the experiments, display a diversity of mechanotransduction properties and biochemical reactions in response to applied mechanical stress, which contributes to the increased susceptibility to hypertrophic scars formation at certain areas of human body characterized by higher skin and muscle tension.

  7. Tissue-engineered skin preserving the potential of epithelial cells to differentiate into hair after grafting.

    Science.gov (United States)

    Larouche, Danielle; Cuffley, Kristine; Paquet, Claudie; Germain, Lucie

    2011-03-01

    The aim of this study was to evaluate whether tissue-engineered skin produced in vitro was able to sustain growth of hair follicles in vitro and after grafting. Different tissues were designed. Dissociated newborn mouse keratinocytes or newborn mouse hair buds (HBs) were added onto dermal constructs consisting of a tissue-engineered cell-derived matrix elaborated from either newborn mouse or adult human fibroblasts cultured with ascorbic acid. After 7-21 days of maturation at the air-liquid interface, no hair was noticed in vitro. Epidermal differentiation was observed in all tissue-engineered skin. However, human fibroblast-derived tissue-engineered dermis (hD) promoted a thicker epidermis than mouse fibroblast-derived tissue-engineered dermis (mD). In association with mD, HBs developed epithelial cyst-like inclusions presenting outer root sheath-like attributes. In contrast, epidermoid cyst-like inclusions lined by a stratified squamous epithelium were present in tissues composed of HBs and hD. After grafting, pilo-sebaceous units formed and hair grew in skin elaborated from HBs cultured 10-26 days submerged in culture medium in association with mD. However, the number of normal hair follicles decreased with longer culture time. This hair-forming capacity after grafting was not observed in tissues composed of hD overlaid with HBs. These results demonstrate that epithelial stem cells can be kept in vitro in a permissive tissue-engineered dermal environment without losing their potential to induce hair growth after grafting.

  8. Skin manifestations in a case of trisomy 16 mosaicism

    DEFF Research Database (Denmark)

    Ousager, Lilian Bomme; Brandrup, Flemming; Andersen, Charlotte Brasch;

    2006-01-01

    We present a 48-year-old man with unilateral dermatological manifestations including hypertrichosis, telangiectasia, hyperkeratosis and hyperpigmentation. Additional findings included skeletal abnormalities and left-sided hearing loss. Skin biopsies showed changes characteristic of porokeratosis........ Fibroblast karyotyping from affected skin demonstrated trisomy 16 mosaicism, in contrast to the normal karyotype in unaffected skin and blood lymphocytes. The possible role of trisomy 16 in porokeratosis is discussed....

  9. Impact of KRAS, BRAF, PIK3CA mutations, PTEN, AREG, EREG expression and skin rash in ≥ 2 line cetuximab-based therapy of colorectal cancer patients.

    Directory of Open Access Journals (Sweden)

    Zacharenia Saridaki

    Full Text Available BACKGROUND: To investigate the predictive significance of KRAS, BRAF, PIK3CA mutational status, AREG- EREG mRNA expression, PTEN protein expression and skin rash in metastatic colorectal cancer (mCRC patients treated with cetuximab containing salvage chemotherapy. METHODS: Primary tumors from 112 mCRC patients were analyzed. The worst skin toxicity during treatment was recorded. RESULTS: KRAS, BRAF and PIK3CA mutations were present in 37 (33%, 8 (7.2% and 11 (9.8% cases, respectively, PTEN was lost in 21 (19.8% cases, AREG and EREG were overexpressed in 48 (45% and 51 (49% cases. In the whole study population, time to tumor progression (TTP and overall survival (OS was significantly lower in patients with KRAS (p = 0.001 and p = 0.026, respectively or BRAF (p = 0.001 and p<0.0001, respectively mutant tumors, downregulation of AREG (p = 0.018 and p = 0.013, respectively or EREG (p = 0.002 and p = 0.004, respectively and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively. In KRAS wt patients TTP and OS was significantly lower in patients with BRAF (p = 0.0001 and p<0.0001, respectively mutant tumors, downregulation of AREG (p = 0.021 and p = 0.004, respectively or EREG (p = 0.0001 and p<0.0001, respectively and grade 0-1 skin rash (p<0.0001 and p<0.0001, respectively. TTP was significantly lower in patients with PIK3CA mutations (p = 0.01 or lost PTEN (p = 0.002. Multivariate analysis revealed KRAS (Hazard Ratio [HR] 4.3, p<0.0001, BRAF mutation (HR: 5.1, p<0.0001, EREG low expression (HR: 1.6, p = 0.021 and absence of severe/moderate skin rash (HR: 4.0, p<0.0001 as independent prognostic factors for decreased TTP. Similarly, KRAS (HR 2.9, p = 0.01, BRAF mutation (HR: 3.0, p = 0.001, EREG low expression (HR: 1.7, p = 0.021, absence of severe/moderate skin rash (HR: 3.7, p<0.0001 and the presence of undifferantited tumours (HR: 2.2, p = 0.001 were revealed as independent prognostic factors for decreased OS. CONCLUSIONS: These results

  10. SKIN AND HAIR CHANGES AFTER FORTY

    Directory of Open Access Journals (Sweden)

    Manisha

    2014-04-01

    Full Text Available Aging is a continuous, dynamic, and an irreversible process. Direct exposure to ultra-violet radiations, skin is particularly prone to early aging, known as photo aging. Skin aging is particularly important because of its visibility and social impact. As women age we will notice changes to our skin and hair during the menopause. Dry, thinning, fragile, less tolerant and sagging skin are common complaints. The main reasons for the change in skin is the loss of estrogen, testosterone and dehydroepiandrosterone (DHEA etc, 1, 2, 3 from the age of 35 onwards up to menopause, the more we have had long-term exposure to the elements, such as sun and wind the more this becomes evident. Estrogen is very involved in the normal function of the skin. It directly affects the function of key cells in the skin, like the fibroblast (produces collagen and elastin, keratinocyte (closely involved in skin protection and melanocytes (involved in evenness of skin color, etc.. It also helps regulate hair follicle function (hair production as well as sebaceous gland activity (producing skin oils. After the age of forty most of women enters menopause, during which estrogens levels decreases, which leads to different types of hair and skin changes which has been described in this article.

  11. Effects of lunar and mars dust simulants on HaCaT keratinocytes and CHO-K1 fibroblasts

    Science.gov (United States)

    Rehders, Maren; Grosshäuser, Bianka B.; Smarandache, Anita; Sadhukhan, Annapurna; Mirastschijski, Ursula; Kempf, Jürgen; Dünne, Matthias; Slenzka, Klaus; Brix, Klaudia

    2011-04-01

    Exposure to lunar dust during Apollo missions resulted in occasional reports of ocular, respiratory and dermal irritations which showed that lunar dust has a risk potential for human health. This is caused by its high reactivity as well as its small size, leading to a wide distribution also inside habitats. Hence, detailed information regarding effects of extraterrestrial lunar dusts on human health is required to best support future missions to moon, mars or other destinations. In this study, we used several methods to assess the specific effects of extraterrestrial dusts onto mammalian skin by exposing HaCaT keratinocytes and CHO-K1 fibroblasts to dusts simulating lunar or mars soils. These particular cell types were chosen because the skin protects the human body from potentially harmful substances and because a well orchestrated program ensures proper wound healing. Keratinocytes and fibroblasts were exposed to the dusts for different durations of time and their effects on morphology and viability of the cells were determined. Cytotoxicity was measured using the MTT assay and by monitoring culture impedance, while phalloidin staining of the actin cytoskeleton was performed to address structural integrity of the cells which was also investigated by propidium iodide intake. It was found that the effects of the two types of dust simulants on the different features of both cell lines varied to a considerable extent. Moreover, proliferation of HaCaT keratinocytes, as analyzed by Ki67 labeling, was suppressed in sub-confluent cultures exposed to lunar dust simulant. Furthermore, experimental evidence is provided for a delay in regeneration of keratinocyte monolayers from scratch-wounding when exposed to lunar dust simulant. The obtained results will facilitate further investigations of dust exposure during wound healing and will ease risk assessment studies e.g., for lunar lander approaches. The investigations will help to determine safety measures to be taken during

  12. Trophic effect of a methanol yeast extract on 3T3 fibroblast cells.

    Science.gov (United States)

    Gallo, Dominique; Dillemans, Monique; Allardin, David; Priem, Fabian; Van Nedervelde, Laurence

    2014-01-01

    With regard to the increase of human life expectancy, interest for topical treatments aimed to counteract skin aging is still growing. Hence, research for bioactive compounds able to stimulate skin fibroblast activity is an attractive topic. Having previously described the effects of a new methanol yeast extract on growth and metabolic activity of Saccharomyces cerevisiae, we studied its effects on 3T3 fibroblasts to evaluate its potential antiaging property. This investigation demonstrates that this extract increases proliferation as well as migration of 3T3 cells and decreases their entrance in senescence and apoptosis phases. Altogether, these results open new perspectives for the formulation of innovative antiaging topical treatments.

  13. Fibroblasts from long-lived rodent species exclude cadmium.

    Science.gov (United States)

    Dostál, Lubomír; Kohler, William M; Penner-Hahn, James E; Miller, Richard A; Fierke, Carol A

    2015-01-01

    Resistance to the lethal effects of cellular stressors, including the toxic heavy metal cadmium (Cd), is characteristic of fibroblast cell lines derived from long-lived bird and rodent species, as well as cell lines from several varieties of long-lived mutant mice. To explore the mechanism of resistance to Cd, we used inductively coupled plasma mass spectroscopy to measure the rate of Cd uptake into primary fibroblasts of 15 rodent species. These data indicate that fibroblasts from long-lived rodent species have slower rates of Cd uptake from the extracellular medium than those from short-lived species. In addition, fibroblasts from short-lived species export more zinc after exposure to extracellular Cd than cells from long-lived species. Lastly, fibroblasts from long-lived rodent species have lower baseline concentrations of two redox-active metals, iron and copper. Our results suggest that evolution of longevity among rodents required adjustment of cellular properties to alter metal homeostasis and to reduce the toxic effects of heavy metals that accumulate over the course of a longer life span.

  14. Development of nanofibrous scaffolds containing gum tragacanth/poly (ε-caprolactone) for application as skin scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Ranjbar-Mohammadi, Marziyeh [Textile Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Bahrami, S. Hajir, E-mail: hajirb@aut.ac.ir [Textile Engineering Department, Amirkabir University of Technology, Tehran (Iran, Islamic Republic of); Center for excellence Modern Textile Characterization, Tehran (Iran, Islamic Republic of)

    2015-03-01

    Outstanding wound healing activity of gum tragacanth (GT) and higher mechanical strength of poly (ε-caprolactone) (PCL) may produce an excellent nanofibrous patch for either skin tissue engineering or wound dressing application. PCL/GT scaffold containing different concentrations of PCL with different blend ratios of GT/PCL was produced using 90% acetic acid as solvent. The results demonstrated that the PCL/GT (3:1.5) with PCL concentration of 20% (w/v) produced nanofibers with proper morphology. Scanning electron microscopy (SEM) and differential scanning calorimetry (DSC) were utilized to characterize the nanofibers. Surface wettability, functional groups analysis, porosity and tensile properties of nanofibers were evaluated. Morphological characterization showed that the addition of GT to PCL solution results in decreasing the average diameter of the PCL/GT nanofibers. However, the hydrophilicity increased in the PCL/GT nanofibers. Slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. PCL/GT nanofibers were used for in vitro cell culture of human fibroblast cell lines AGO and NIH 3T3 fibroblast cells. MTT assay and SEM results showed that the biocomposite PCL/GT mats enhanced the fibroblast adhesion and proliferation compared to PCL scaffolds. The antibacterial activity of PCL/GT and GT nanofibers against Staphylococcus aureus and Pseudomonas aeruginosa was also examined. - Highlights: • A new skin tissue engineering scaffold from poly (ε-caprolactone) (PCL) and gum tragacanth (GT) has been developed. • These scaffolds might be an effectual simulator of the structure and composition of native skin. • Very slight increase in melting peaks was observed due to the blending of PCL with GT nanofibers. • Biodegradation, water uptake and hydrophilicity properties of these scaffolds showed that produced scaffolds were adherent. • The electrospun PCL/GT scaffold can promote the skin regeneration of full

  15. Adipose Tissue-Derived Stromal Cells Inhibit TGF-beta 1-Induced Differentiation of Human Dermal Fibroblasts and Keloid Scar-Derived Fibroblasts in a Paracrine Fashion

    NARCIS (Netherlands)

    Spiekman, Maroesjka; Przybyt, Ewa; Plantinga, Josee A.; Gibbs, Susan; van der Lei, Berend; Harmsen, Martin C.

    2014-01-01

    Background: Adipose tissue-derived stromal cells augment wound healing and skin regeneration. It is unknown whether and how they can also influence dermal scarring. The authors hypothesized that adipose tissue-derived stromal cells inhibit adverse differentiation of dermal fibroblasts induced by the

  16. Irradiated fibroblasts promote epithelial–mesenchymal transition and HDGF expression of esophageal squamous cell carcinoma

    Energy Technology Data Exchange (ETDEWEB)

    Bao, Ci-Hang; Wang, Xin-Tong [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China); Ma, Wei [Department of Radiation Oncology, Cancer Hospital, Genaral Hospital of Ningxia Medical University, Yinchuan 750000 (China); Wang, Na-Na; Nesa, Effat un; Wang, Jian-Bo; Wang, Cong; Jia, Yi-Bin; Wang, Kai [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China); Tian, Hui [Department of Thoracic Surgery, Qilu Hospital of Shandong University, Jinan 250012 (China); Cheng, Yu-Feng, E-mail: qlcyf1965@126.com [Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan 250012 (China)

    2015-03-06

    Recent evidence suggested that nonirradiated cancer-associated fibroblasts (CAFs) promoted aggressive phenotypes of cancer cells through epithelial–mesenchymal transition (EMT). Hepatoma-derived growth factor (HDGF) is a radiosensitive gene of esophageal squamous cell carcinoma (ESCC). This study aimed to investigate the effect of irradiated fibroblasts on EMT and HDGF expression of ESCC. Our study demonstrated that coculture with nonirradiated fibroblasts significantly increased the invasive ability of ESCC cells and the increased invasiveness was further accelerated when they were cocultured with irradiated fibroblasts. Scattering of ESCC cells was also accelerated by the supernatant from irradiated fibroblasts. Exposure of ESCC cells to supernatant from irradiated fibroblasts resulted in decreased E-cadherin, increased vimentin in vitro and β-catenin was demonstrated to localize to the nucleus in tumor cells with irradiated fibroblasts in vivo models. The expression of HDGF and β-catenin were increased in both fibroblasts and ESCC cells of irradiated group in vitro and in vivo models. Interestingly, the tumor cells adjoining the stromal fibroblasts displayed strong nuclear HDGF immunoreactivity, which suggested the occurrence of a paracrine effect of fibroblasts on HDGF expression. These data suggested that irradiated fibroblasts promoted invasion, growth, EMT and HDGF expression of ESCC. - Highlights: • Irradiated CAFs accelerated invasiveness and scattering of ESCC cell lines. • Irradiated CAFs promoted EMT of ESCC cells. • Irradiated fibroblasts induced nuclear β-catenin relocalization in ESCC cells. • Irradiated fibroblasts increased HDGF expression in vitro and in vivo.

  17. 尼纶线、薇荞线对缝合部位皮肤衰老因子影响的实验研究%Experimental study for comparing the effects of nylon line and vicryl line on the skin aging factors on the operation area

    Institute of Scientific and Technical Information of China (English)

    马恬; 贾赤宇; 张辉; 徐凯

    2011-01-01

    Objective To explore a better clinical surgical suture by comparing the effect of nylon line and vicryl line on the skin aging on the operation area. Methods Twenty aging rabbits were randomly divided into 2 groups nnylon group and vicryl group. Two different kinds of lines were implanted under the skin of rabbits at day 1 ,30,45 ,60 , 75 , 83 , 87 , respectively. At day 90 , skin samples from surgical areas were cut and divided irUo two pafls in two goups. One part of samples was used to determine total antioxidant capacity ( T AOC) ,superoxide dismutase ( SOD) activity. the contents of hydrogen peroxide ( H202 ) and hydroxyproline( HYP) in aging skins,while the other part was used to observe the skin histopathological changes on the operation area. Results The total antioxidanc capacity,the superoxide dismutase capacity.and the hydroxyproline content in vicryl group were significantly higher than those in nylon group( P < 0. 05 ) . The level of hydrogen peroxide was significantly lower in vicryl group than that in nylon group( P< 0. 05 ) . Conclusion Compared with the nylon line,rhe vicryl line can delay the skin aging hy increasing the total antioxidant capacity and superoxide dismutase. activity and decreasing the hydrogen peroxide in the implantation site. Compared wiLh the nylon line, the vicryl line may be more favorable for wound healing by regulating the expression of skin aging factors in the operation region.%目的 建立家兔的衰老模型,通过比较尼龙线和薇荞线对于手术区局部皮肤衰老因子影响的研究,以此来指导临床选择缝线. 方法 20只衰老家兔随机分为尼龙线组和薇荞线组.将两种线植入家兔皮肤下,分别于3 d,7 d,15 d,30 d,45 d,60 d,90 d切取各缝线手术区皮肤样本,一份测定家兔皮肤组织中总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)活性、过氧化氢(H2O2)及羟脯氨酸的含量,另一份做HE染色,观察手术区皮肤组织学的改变. 结果 薇荞

  18. Effect of a monoclonal antibody against fibroblast growth factor 10 in a guinea pig model of psoriasis-like skin inflammation%成纤维细胞生长因子10单克隆抗体对豚鼠银屑病样模型的作用研究

    Institute of Scientific and Technical Information of China (English)

    梅向林; 夏建新; 牟妍; 王敬医; 李雪; 朱文静; 李福秋; 金仙花; 于凯

    2013-01-01

    Objective To evaluate the therapeutic effect of local application of an anti-fibroblast growth factor 10 (FGF10) monoclonal antibody (MoAb) on a guinea pig model of psoriasis-like skin inflammation.Methods A model of psoriasis-like skin inflammation was established by applying 5% propranolol hydrochloride emulsion to the dorsal skin of ears of 45 guinea pigs,which were then classified into 5 groups:model group receiving no treatment,hydrocortisone butyrate group,high-,medium-and low-dose MoAb groups receiving topical treatment with hydrocortisone butyrate,anti-FGF10 MoAb of 0.188 g/L,0.094 g/L and 0.063 g/L,respectively,twice daily.Nine guinea pigs receiving no propranolol challenge nor treatment served as the blank control group.The response of guinea pigs to propranolol hydrochloride emulsion and therapeutic agents,such as scratch behavior,hair status and skin status,was observed and recorded.After two weeks of treatment,all the guinea pigs were sacrificed,and skin samples were resected from the ears followed by the evaluation of Baker score,measurement of epidermal thickness,and enumeration of mononuclear cells.SPSS 13.0 software was used for data processing.Baker score was compared by rank sum test,mononuclear cell count and epidermal thickness by analysis of variance.Pairwise comparisons were carried out by the least significant difference (LSD) procedure.Results The number of mononuclear cells was similar between the high-dose MoAb group and blank control group (t =0.77,P > 0.05),but higher in the other treatment groups than in the high-dose MoAb group and blank control group (all P < 0.05).Increased epidermal thickness was observed in the three MoAb groups compared with the hydrocortisone butyrate group (all P < 0.05),while there was no statistical difference between the three MoAb groups (all P > 0.05).Conclusions The anti-FGF10 MoAb obviously attenuates the pathological changes,affects the proliferation of keratinocytes,and markedly suppresses

  19. Generation of iPS cell lines from schizophrenia patients using a non-integrative method

    Directory of Open Access Journals (Sweden)

    Jaroslaw Sochacki

    2016-07-01

    Full Text Available Skin biopsies were collected from three male patients (age 45, 44 and 44 with clinically diagnosed schizophrenia. The patients were diagnosed according to DSM-5 criteria by a trained psychiatrist. Dermal fibroblast cell lines were established and expanded for subsequent reprogramming procedures. Induced pluripotent stem (iPS cells were derived using the integration-free CytoTune®-iPS 2.0 Sendai Reprogramming Kit, containing Sendai virus particles of the four Yamanaka factors Oct3/4, Sox2, Klf4 and c-Myc.

  20. Do you know this syndrome? Dyspigmentation along the Blaschko lines caused by trisomy 7 mosaicism*

    Science.gov (United States)

    de Gouveia, Miguel Pinto; Coutinho, Inês; Teixeira, Vera; d'Oliveira, Renata; Venâncio, Margarida; Moreno, Ana

    2016-01-01

    Dyspigmentation along the Blaschko lines is strongly suggestive of a mosaic skin disorder. We report a 9-year-old male patient who presented with swirls and streaks of both hypo and hyperpigmentation involving the entire body. Additionally, he had hypertrichosis, musculoskeletal and minor neurodevelopment abnormalities but no intellectual disability. Cultured fibroblast displayed trisomy 7 mosaicism, which can explain this pigmentary phenotype. Widespread dyspigmentation associated with involvement of other organs should prompt systemic examination to detect additional anomalies and genetic evaluation should be considered, even with normal fetal karyotype. PMID:28099615

  1. Antioxidant effects of the sarsaparilla via scavenging of reactive oxygen species and induction of antioxidant enzymes in human dermal fibroblasts.

    Science.gov (United States)

    Park, Gunhyuk; Kim, Tae-mi; Kim, Jeong Hee; Oh, Myung Sook

    2014-07-01

    Ultraviolet (UV) radiation from sunlight causes distinct changes in collagenous skin tissues as a result of the breakdown of collagen, a major component of the extracellular matrix. UV irradiation downregulates reactive oxygen species (ROS)-elimination pathways, thereby promoting the production of ROS, which are implicated in skin aging. Smilax glabra Roxb (sarsaparilla) has been used in folk medicine because of its many effects. However, no study on the protective effects of sarsaparilla root (SR) on human dermal fibroblasts has been reported previously. Here, we investigated the protective effect of SR against oxidative stress in dermal fibroblasts. SR significantly inhibited oxidative damage and skin-aging factor via mitogen-activated protein kinase signaling pathways. Also, SR decreased Ca(2+) and ROS, mitochondrial membrane potential, dysfunction, and increased glutathione, NAD(P)H dehydrogenase and heme oxygenase-1. These results demonstrate that SR can protect dermal fibroblasts against UVB-induced skin aging via antioxidant effects.

  2. Glutathione metabolism in the HaCaT cell line as a model for the detoxification of the model sensitisers 2,4-dinitrohalobenzenes in human skin.

    Science.gov (United States)

    Jacquoilleot, Sandrine; Sheffield, David; Olayanju, Adedamola; Sison-Young, Rowena; Kitteringham, Neil R; Naisbitt, Dean J; Aleksic, Maja

    2015-08-19

    Glutathione (GSH) is the most prominent antioxidant in cells and the co-factor of an important set of enzymes involved in the skin metabolic clearance system, glutathione S-transferases (GST). Here, we describe an LC-MS (liquid chromatography-mass spectroscopy) method to measure GSH and its disulfide form (GSSG) in HaCaT cells and a 3D Reconstructed Human Epidermis (RHE) model. In our assay, the basal level of GSH in both systems was in the low nmol/mg soluble protein range, while the level of GSSG was systematically below our limit of quantification (0.1 μM). We found that 2,4-dinitrohalobenzenes deplete the GSH present in HaCaT cells within the first hour of exposure, in a dose dependent manner. The level of GSH in HaCaT cells treated with a single non-toxic dose of 10 μM of dinitrohalobenzene was also shown to increase after two hours. While cells treated with 1-chloro-2,4-dinitrobenzene (DNCB) and 1-fluoro-2,4-dinitrobenzene (DNFB) repleted GSH to levels similar to untreated control cells within 24h, 1-bromo-2,4-dinitrobenzene (DNBB) seemed to prevent such a repletion and appeared to be the most toxic compound in all assays. A mathematical modelling of experimental results was performed to further rationalise the differences observed between test chemicals. For this purpose the biological phenomena observed were simplified into two sequential events: the initial depletion of the GSH stock after chemical treatment followed by the repletion of the GSH once the chemical was cleared. Activation of the nuclear factor E2-related factor 2 (Nrf2) pathway was observed with all compounds within two hours, and at concentrations less than 10 μM. These data show that GSH depletion and repletion occur rapidly in skin cells and emphasize the importance of conducting kinetic studies when performing in vitro experiments exploring skin sensitization.

  3. Altered chromosome 6 in immortal human fibroblasts.

    Science.gov (United States)

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  4. Altered chromosome 6 in immortal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.; Ozer, H.L. (New Jersey Medical School, Newark, NJ (United States)); Patsalis, P.; Henderson, A.S. (City Univ. of New York, NY (United States))

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. 66 refs., 6 figs., 2 tabs.

  5. Your Skin

    Science.gov (United States)

    ... your body. Without skin, people's muscles, bones, and organs would be hanging out all over the place. Skin holds everything together. It also: protects our bodies helps keep our bodies at just the right temperature allows us to have the sense of touch Don't Miss Your Epidermis The ...

  6. Oily skin

    Science.gov (United States)

    ... keep your skin clean using warm water and soap, or a soapless cleanser. Clean your face with astringent pads if frequent face washing causes irritation. Use only water-based or oil-free cosmetics if you have oily skin. Your ...

  7. Tattoo ink nanoparticles in skin tissue and fibroblasts

    OpenAIRE

    2015-01-01

    Tattooing has long been practised in various societies all around the world and is becoming increasingly common and widespread in the West. Tattoo ink suspensions unquestionably contain pigments composed of nanoparticles, i.e., particles of sub-100 nm dimensions. It is widely acknowledged that nanoparticles have higher levels of chemical activity than their larger particle equivalents. However, assessment of the toxicity of tattoo inks has been the subject of little research and ink manufactu...

  8. The effect of the anti-allergic agent avil on abnormal scar fibroblasts.

    Science.gov (United States)

    Venugopal, J; Ramakrishnan, M; Habibullah, C M; Babu, M

    1999-05-01

    Abnormal wound healing in humans leads to the formation of hypertrophic scar and keloids. These abnormal scars accumulate excessive extracellular matrix proteins through increased synthesis as well as decreased degradation. In order to find a therapeutic control for scar formation, we investigated the effect of avil (pheniramine maleate) on fibroblasts cultured from abnormal scars in comparison to normal skin. We observed a decrease in the proliferation rate in cells from normal skin (39%), hypertrophic scar (44%), keloid (63%) and in DNA synthesis in cells from normal skin (50%), hypertrophic scar (55%) and keloid (63%) treated with 8 mM avil (72 h). The rate of decrease in collagen synthesis in normal skin (44%), hypertrophic scar (74%) and keloid fibroblast (73%) correlated with changes in DNA synthesis.

  9. Smac/DIABLO regulates the apoptosis of hypertrophic scar fibroblasts.

    Science.gov (United States)

    Liu, Bao-Heng; Chen, Liang; Li, Shi-Rong; Wang, Zhen-Xiang; Cheng, Wen-Guang

    2013-09-01

    In abnormal skin wound healing, hypertrophic scars (HS) are characterized by excessive fibroblast hypercellularity and an overproduction of collagen, leading to atypical extracellular matrix (ECM) remodeling. Although the exact mechanisms of HS remain unclear, decreased HS fibroblast (HSFB) apoptosis and increased proliferation are evident in the development of HS. In this study, the contribution of the second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein (IAP)-binding protein with a low isoelectric point (pI) (Smac/DIABLO), an apoptosis-promoting protein released from the mitochondria, was investigated in human normal skin and HSFB cultures. The expression of Smac/DIABLO is usually decreased in many malignant tumors compared with normal tissues. Immunohistochemical analysis of skin tissues and the western blot analyses of fibroblasts revealed that the expression of Smac/DIABLO was lower in HS tissues compared with normal skin tissues. Of note, adenovirus-mediated Smac/DIABLO overexpression in the cultured HSFBs significantly reduced cell proliferation, as detected by the cell counting kit-8, and increased caspase-3 and -9 activity, as detected by spectrofluorimetry. In addition, it increased apoptosis, as detected by fluorescence-activated cell sorting (FACS). Furthermore, we found that the silencing of Smac with siRNA in the HSFBs induced a noticeable decrease in caspase-3 and -9 activity, leading to a significant reduction in apoptosis. In addition, the mRNA expression of type I and III pro-collagen detected in the HSFBs was significantly increased following the silencing of Smac with siRNA and was inhibited following Smac/DIABLO overexpression, as shown by real-time RT-PCR. In conclusion, Smac/DIABLO decreases the proliferation and increases the apoptosis of HSFBs. To our knowledge, the data from our study suggest for the first time that Smac/DIABLO is a novel therapeutic target for HS.

  10. PED/PEA-15 controls fibroblast motility and wound closure by ERK1/2-dependent mechanisms.

    Science.gov (United States)

    Buonomo, Roberta; Giacco, Ferdinando; Vasaturo, Angela; Caserta, Sergio; Guido, Stefano; Pagliara, Valentina; Garbi, Corrado; Mansueto, Gelsomina; Cassese, Angela; Perruolo, Giuseppe; Oriente, Francesco; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2012-05-01

    Cell migration is dependent on the control of signaling events that play significant roles in creating contractile force and in contributing to wound closure. We evaluated wound closure in fibroblasts from mice overexpressing (TgPED) or lacking ped/pea-15 (KO), a gene overexpressed in patients with type 2 diabetes. Cultured skin fibroblasts isolated from TgPED mice showed a significant reduction in the ability to recolonize wounded area during scratch assay, compared to control fibroblasts. This difference was observed both in the absence and in the presence of mytomicin C, an inhibitor of mitosis. In time-lapse experiments, TgPED fibroblasts displayed about twofold lower velocity and diffusion coefficient, as compared to controls. These changes were accompanied by reduced spreading and decreased formation of stress fibers and focal adhesion plaques. At the molecular level, TgPED fibroblasts displayed decreased RhoA activation and increased abundance of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2). Inhibition of ERK1/2 activity by PD98059 restored RhoA activation, cytoskeleton organization and cell motility, and almost completely rescued wound closure of TgPED fibroblasts. Interestingly, skin fibroblasts isolated from KO mice displayed an increased wound closure ability. In vivo, healing of dorsal wounds was delayed in TgPED and accelerated in KO mice. Thus, PED/PEA-15 may affect fibroblast motility by a mechanism, at least in part, mediated by ERK1/2.

  11. Antioxidant and potential anti-inflammatory activity of extracts and formulations of white tea, rose, and witch hazel on primary human dermal fibroblast cells

    OpenAIRE

    Hili Pauline; Thring Tamsyn SA; Naughton Declan P

    2011-01-01

    Abstract Background Numerous reports have identified therapeutic roles for plants and their extracts and constituents. The aim of this study was to assess the efficacies of three plant extracts for their potential antioxidant and anti-inflammatory activity in primary human skin fibroblasts. Methods Aqueous extracts and formulations of white tea, witch hazel and rose were subjected to assays to measure anti-collagenase, anti-elastase, trolox equivalent and catalase activities. Skin fibroblast ...

  12. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation.

    Directory of Open Access Journals (Sweden)

    Qi-cui Du

    Full Text Available Hypertrophic scars (HTS, the excessive deposition of scar tissue by fibroblasts, is one of the most common skin disorders. Fibroblasts derived from surgical scar tissue produce high levels of α-smooth muscle actin (α-SMA and transforming growth factor-β1 (TGF-β1. However, the molecular mechanisms for this phenomenon is poorly understood. Thus, the purpose of this study was to evaluate the molecular mechanisms of HTS and their potential therapeutic implications. Fibroblasts derived from skin HTS were cultured and characterized in vitro. The fibroblasts were synchronized and randomly assigned to two groups: cyclic stretch and cyclic stretch pre-treated with SB203580 (a p38MAPK inhibitor. Cyclic stretch at 10% strain was applied at a loading frequency of 10 cycles per minute (i.e. 5 seconds of tension and 5 seconds of relaxation for 0 h, 6 h and 12 h. Cyclic stretch on HTS fibroblasts led to an increase in the expression of α-SMA and TGF-β1 mRNA and protein and the phosphorylation of p38MAPK. SB203580 reversed these effects and caused a decrease in matrix contraction. Furthermore, HTS fibroblast growth was partially blocked by p38MAPK inhibition. Therefore, the mechanism of cyclic stretch involves p38 MAPK, and its inhibition is suggested as a novel therapeutic strategy for HTS.

  13. The effect of p38MAPK on cyclic stretch in human facial hypertrophic scar fibroblast differentiation.

    Science.gov (United States)

    Du, Qi-cui; Zhang, Dai-zun; Chen, Xiu-juan; Lan-Sun, Gui; Wu, Min; Xiao, Wen-lin

    2013-01-01

    Hypertrophic scars (HTS), the excessive deposition of scar tissue by fibroblasts, is one of the most common skin disorders. Fibroblasts derived from surgical scar tissue produce high levels of α-smooth muscle actin (α-SMA) and transforming growth factor-β1 (TGF-β1). However, the molecular mechanisms for this phenomenon is poorly understood. Thus, the purpose of this study was to evaluate the molecular mechanisms of HTS and their potential therapeutic implications. Fibroblasts derived from skin HTS were cultured and characterized in vitro. The fibroblasts were synchronized and randomly assigned to two groups: cyclic stretch and cyclic stretch pre-treated with SB203580 (a p38MAPK inhibitor). Cyclic stretch at 10% strain was applied at a loading frequency of 10 cycles per minute (i.e. 5 seconds of tension and 5 seconds of relaxation) for 0 h, 6 h and 12 h. Cyclic stretch on HTS fibroblasts led to an increase in the expression of α-SMA and TGF-β1 mRNA and protein and the phosphorylation of p38MAPK. SB203580 reversed these effects and caused a decrease in matrix contraction. Furthermore, HTS fibroblast growth was partially blocked by p38MAPK inhibition. Therefore, the mechanism of cyclic stretch involves p38 MAPK, and its inhibition is suggested as a novel therapeutic strategy for HTS.

  14. Effects of different concentrations of putrescine on proliferation, migration and apoptosis of human skin fibroblasts%不同浓度腐胺对人皮肤成纤维细胞增殖、迁移和凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    陈健霞; 荣新洲; 樊桂成; 李松泽; 李庆辉

    2015-01-01

    目的:探讨不同浓度腐胺对人皮肤成纤维细胞(human skin fibroblasts,HSF)增殖、迁移、凋亡的影响。方法将体外HSF分为腐胺组和对照组,以含腐胺浓度分别为0.5、1、5、10、50、100、500、1000μg/ml完全培养基培养细胞设为腐胺组,不添加腐胺设为对照组。经对照组及不同浓度腐胺组处理的HSF培养24 h后,再以四唑化合物电子耦联显色法(MTS法)测定细胞增殖能力(用吸光度值表示),Transwell迁移实验测定细胞的迁移能力,流式细胞技术(Flow Cytometry, FCM)Annexin V/PI标记法测定细胞凋亡率。结果(1)细胞增殖能力(MTS)测定结果显示,0.5、1、5、10μg/ml腐胺组HSF的吸光度值较对照组升高(P0.05);(2)Transwell迁移实验结果显示,1μg/ml腐胺组穿过微孔膜的细胞数目较对照组显著增加(P0.05);(3)流式细胞凋亡结果显示,0.5、1、5、10μg/ml腐胺组HSF凋亡率较对照组显著降低(P0.05)。结论微量腐胺可维持细胞正常的迁移能力,显著提高HSF的细胞增殖能力,而较高浓度的腐胺能显著抑制HSF迁移与增殖,并诱导细胞发生凋亡,提示不同浓度的腐胺会对创面愈合起到完全不同的作用。%Objective To explore the effects of different concentrations of putrescine on the proliferation, migration and apoptosis of human skin fibroblasts (HSF). Methods HSF cultured in the presence of 0.5, 1.0, 5.0, 10, 50, 100, 500, and 1000μg/ml putrescine for 24 h were examined for the changes in the cell proliferation, migration, and apoptosis using MTS assay, Transwell migration assay, and flow cytometry, respectively. Results Compared with the control cells, HSF cultured with 0.5, 1.0, 5.0, and 10μg/ml putrescine showed significantly increased cell proliferation (P0.05). Putrescine at 1 μg/ml most significantly enhanced the cell migration (P0.05). HSF treated with 0.5, 1.0, 5.0, and 10

  15. 过氧化物酶体增殖物激活受体γ激动剂拮抗人皮肤成纤维细胞转化生长因子β1机制研究%Mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ agonists on transforming growth factor β1 in adult skin fibroblasts

    Institute of Scientific and Technical Information of China (English)

    杨崇志; 张会堂; 王公升; 周海全; 马驰; 胡大海

    2010-01-01

    目的 了解过氧化物酶体增殖物激活受体γ(PPARγ)激动剂拈抗TGF-β1促皮肤瘢痕化的效应机制.方法 体外培养正常成人皮肤Fb,根据培养基中所加刺激物不同分为空白对照组(无血清DMEM培养基),TGF-β1组(DMEM培养基加入含终浓度10 ng/mL TGF-β1作用48 h),曲格列酮组(DMEM培养基加入含终浓度10μmol/L曲格列酮作用2 h,再加入10 ng/mL TGF-β1作用48h),15-脱氧前列腺素J2(15d-PGJ2)组(DMEM培养基加入含终浓度10 μmol/L 15d-PGJ2作用2 h,再加入10 ng/mL TGF-β1作用48 h).应用蛋白质印迹法检测结缔组织生长因子(CTGF)的表达,实时荧光RT-PCR检测CTGF、基质金属蛋白酶1(MMP-1)及血小板源性生长因子(PDGF)的mRNA水平变化.对数据进行单因素方差分析.结果 TGF-β1组的CTGF蛋白和mRNA表达水平均高于空白对照组,曲格列酮组和15d-PGJ2组的CTGF蛋白及mRNA表达水平低于TGF-β1组.空白对照组、TGF-β1组、曲格列酮组、15d-PGJ 2组MMP-1 mRNA表达水平分别为1.281±0.195、0.193±0.051、0.417±0.043、0.485±0.027,其中TGF-β1组低于空白对照组(F=12.811,P<0.01),曲格列酮组、15d-PGJ2组高于TGF-β1组(F=12.811,P值均小于0.01).空白对照组、TGF-β1组、曲格列酮组、15d-PGJ2组PDGF mRNA表达水平分别为0.349±0.057、1.044±0.237、0.677±0.055、0.511±0.017,其中TGF-β1组高于空白对照组(F=16.848,P<0.01),曲格列酮组、15d-PGJ2组低于TGF-β1组(F=16.848,P值均小于0.01).结论 激活后的PPARγ对TGF-β1下游反应因子CTGF的抑制作用,可能是其拮抗TGF-β1促皮肤瘢痕化的主要机制,而对细胞因子MMP-1、PDGF的影响可能也是其机制之一.%Objective To study the mechanism underlying the inhibitory effects of peroxisome proliferator-activated receptor γ (PPARγ) agonists on transforming growth factor β 1 (TGF-β1)-induced scarring of skin. Methods Fibroblasts isolated from healthy adult skin were cultured in vitro and divided into blank

  16. Fibroblasts and myofibroblasts in wound healing

    Directory of Open Access Journals (Sweden)

    Darby IA

    2014-11-01

    Full Text Available Ian A Darby,1 Betty Laverdet,2 Frédéric Bonté3, Alexis Desmoulière2 1School of Medical Sciences, RMIT University, Melbourne, VIC, Australia; 2Department of Physiology and EA 6309, FR 3503, Faculties of Medicine and Pharmacy, University of Limoges, Limoges, France; 3LVMH Recherche, Saint Jean de Braye, France Abstract: (Myofibroblasts are key players for maintaining skin homeostasis and for orchestrating physiological tissue repair. (Myofibroblasts are embedded in a sophisticated extracellular matrix (ECM that they secrete, and a complex and interactive dialogue exists between (myofibroblasts and their microenvironment. In addition to the secretion of the ECM, (myofibroblasts, by secreting matrix metalloproteinases and tissue inhibitors of metalloproteinases, are able to remodel this ECM. (Myofibroblasts and their microenvironment form an evolving network during tissue repair, with reciprocal actions leading to cell differentiation, proliferation, quiescence, or apoptosis, and actions on growth factor bioavailability by binding, sequestration, and activation. In addition, the (myofibroblast phenotype is regulated by mechanical stresses to which they are subjected and thus by mechanical signaling. In pathological situations (excessive scarring or fibrosis, or during aging, this dialogue between the (myofibroblasts and their microenvironment may be altered or disrupted, leading to repair defects or to injuries with damaged and/or cosmetic skin alterations such as wrinkle development. The intimate dialogue between the (myofibroblasts and their microenvironment therefore represents a fascinating domain that must be better understood in order not only to characterize new therapeutic targets and drugs able to prevent or treat pathological developments but also to interfere with skin alterations observed during normal aging or premature aging induced by a deleterious environment. Keywords: myofibroblast, fibroblast, α-smooth muscle actin

  17. Examining the Genomic Influence of Skin Antioxidants In Vitro

    Directory of Open Access Journals (Sweden)

    James V. Gruber

    2010-01-01

    Full Text Available A series of well-known, purified antioxidants including: Resveratrol, Epigallocatechin Gallate (EGCG, Genistein, Rosavin, Puerarin, Chlorogenic Acid, Propolis and two newer unexplored isoflavonoids isolated from Maclura pomifera (Osage Orange including Pomiferin and Osajin, were applied to Normal Human Dermal Fibroblasts (NHDF and Normal Human Dermal Keratinocytes (NHEK for 24 hours. The resulting treated cells were then examined using human gene microarrays supplied by Agilent. These chips typically have somewhere on the order of 30,000 individual genes which are expressed in the human genome. For our study, this large list of genes was reduced to 205 principal genes thought to be important for skin and each individual ingredient was examined for its influence on the culled list of genes. Working on a hypothesis that there may be some common genes which are either upregulated or downregulated by all or most of these ingredients, a short list of genes for each cell line was developed. What appears to emerge from these studies is that several genes in the gene pool that was screened are influenced by most or all of the molecules of interest. Genes that appear to be upregulated in both cell lines by all the ingredients include: ACLY, AQP3, COX1, NOS3, and PLOD3. Genes that appear to be downregulated in both cell lines by all ingredients include only PGR.

  18. Examining the genomic influence of skin antioxidants in vitro.

    Science.gov (United States)

    Gruber, James V; Holtz, Robert

    2010-01-01

    A series of well-known, purified antioxidants including: Resveratrol, Epigallocatechin Gallate (EGCG), Genistein, Rosavin, Puerarin, Chlorogenic Acid, Propolis and two newer unexplored isoflavonoids isolated from Maclura pomifera (Osage Orange) including Pomiferin and Osajin, were applied to Normal Human Dermal Fibroblasts (NHDF) and Normal Human Dermal Keratinocytes (NHEK) for 24 hours. The resulting treated cells were then examined using human gene microarrays supplied by Agilent. These chips typically have somewhere on the order of 30,000 individual genes which are expressed in the human genome. For our study, this large list of genes was reduced to 205 principal genes thought to be important for skin and each individual ingredient was examined for its influence on the culled list of genes. Working on a hypothesis that there may be some common genes which are either upregulated or downregulated by all or most of these ingredients, a short list of genes for each cell line was developed. What appears to emerge from these studies is that several genes in the gene pool that was screened are influenced by most or all of the molecules of interest. Genes that appear to be upregulated in both cell lines by all the ingredients include: ACLY, AQP3, COX1, NOS3, and PLOD3. Genes that appear to be downregulated in both cell lines by all ingredients include only PGR.

  19. Role of Age-Associated Alterations of the Dermal Extracellular Matrix Microenvironment in Human Skin Aging

    OpenAIRE

    Quan, Taihao; Fisher, Gary J.

    2015-01-01

    Human skin is largely composed of a collagen-rich connective tissue, which provides structural and functional support. The collagen-rich connective tissue is produced, organized, and maintained by dermal fibroblasts. During aging, dermal collagen fibrils undergo progressive loss and fragmentation, leading to thin and structurally weakened skin. Age-related alterations of collagen fibrils impairs skin structure and function and creates a tissue microenvironment that promotes age-related skin d...

  20. Race Does Not Predict Melanocyte Heterogeneous Responses to Dermal Fibroblast-Derived Mediators.

    Directory of Open Access Journals (Sweden)

    Pornthep Sirimahachaiyakul

    Full Text Available Abnormal pigmentation following cutaneous injury causes significant patient distress and represents a barrier to recovery. Wound depth and patient characteristics influence scar pigmentation. However, we know little about the pathophysiology leading to hyperpigmentation in healed shallow wounds and hypopigmentation in deep dermal wound scars. We sought to determine whether dermal fibroblast signaling influences melanocyte responses.Epidermal melanocytes from three Caucasians and three African-Americans were genotyped for single nucleotide polymorphisms (SNPs across the entire genome. Melanocyte genetic profiles were determined using principal component analysis. We assessed melanocyte phenotype and gene expression in response to dermal fibroblast-conditioned medium and determined potential mesenchymal mediators by proteome profiling the fibroblast-conditioned medium.Six melanocyte samples demonstrated significant variability in phenotype and gene expression at baseline and in response to fibroblast-conditioned medium. Genetic profiling for SNPs in receptors for 13 identified soluble fibroblast-secreted mediators demonstrated considerable heterogeneity, potentially explaining the variable melanocyte responses to fibroblast-conditioned medium.Our data suggest that melanocytes respond to dermal fibroblast-derived mediators independent of keratinocytes and raise the possibility that mesenchymal-epidermal interactions influence skin pigmentation during cutaneous scarring.

  1. Effect of fibroblast-seeded artificial dermis on wound healing.

    Science.gov (United States)

    Jang, Joon Chul; Choi, Rak-Jun; Han, Seung-Kyu; Jeong, Seong-Ho; Kim, Woo-Kyung

    2015-04-01

    In covering wounds, efforts should include use of the safest and least invasive methods with a goal of achieving optimal functional and cosmetic outcome. The recent development of advanced technology in wound healing has triggered the use of cells and/or biological dermis to improve wound healing conditions. The purpose of the study was to evaluate the effects of fibroblast-seeded artificial dermis on wound healing efficacy.Ten nude mice were used in this study. Four full-thickness 6-mm punch wounds were created on the dorsal surface of each mouse (total, 40 wounds). The wounds were randomly assigned to one of the following 4 treatments: topical application of Dulbecco phosphate-buffered saline (control), human fibroblasts (FB), artificial dermis (AD), and human fibroblast-seeded artificial dermis (AD with FB). On the 14th day after treatment, wound healing rate and wound contraction, which are the 2 main factors determining wound healing efficacy, were evaluated using a stereoimage optical topometer system, histomorphological analysis, and immunohistochemistry.The results of the stereoimage optical topometer system demonstrated that the FB group did not have significant influence on wound healing rate and wound contraction. The AD group showed reduced wound contraction, but wound healing was delayed. The AD with FB group showed decreased wound contraction without significantly delayed wound healing. Histomorphological analysis exhibited that more normal skin structure was regenerated in the AD with FB group. Immunohistochemistry demonstrated that the AD group and the AD with FB group produced less α-smooth muscle actin than the control group, but this was not shown in the FB group.Fibroblast-seeded artificial dermis may minimize wound contraction without significantly delaying wound healing in the treatment of skin and soft tissue defects.

  2. A Luciferase-Expressing Leishmania braziliensis Line That Leads to Sustained Skin Lesions in BALB/c Mice and Allows Monitoring of Miltefosine Treatment Outcome.

    Directory of Open Access Journals (Sweden)

    Adriano C Coelho

    2016-05-01

    Full Text Available Leishmania braziliensis is the most prevalent species isolated from patients displaying cutaneous and muco-cutaneous leishmaniasis in South America. However, there are difficulties for studying L. braziliensis pathogenesis or response to chemotherapy in vivo due to the natural resistance of most mouse strains to infection with these parasites. The aim of this work was to develop an experimental set up that could be used to assess drug efficacy against L. braziliensis. The model was tested using miltefosine.A L. braziliensis line, originally isolated from a cutaneous leishmaniasis patient, was passaged repeatedly in laboratory rodents and further genetically manipulated to express luciferase. Once collected from a culture of parasites freshly transformed from amastigotes, 106 wild type or luciferase-expressing stationary phase promastigotes were inoculated subcutaneously in young BALB/c mice or golden hamsters. In both groups, sustained cutaneous lesions developed at the site of inoculation, no spontaneous self- healing being observed 4 months post-inoculation, if left untreated. Compared to the wild type line features, no difference was noted for the luciferase-transgenic line. Infected animals were treated with 5 or 15 mg/kg/day miltefosine orally for 15 days. At the end of treatment, lesions had regressed and parasites were not detected. However, relapses were observed in animals treated with both doses of miltefosine.Here we described experimental settings for a late-healing model of cutaneous leishmaniasis upon inoculation of a luciferase-expressing L. braziliensis line that can be applied to drug development projects. These settings allowed the monitoring of the transient efficacy of a short-term miltefosine administration.

  3. Skin Cancer Prevention

    Science.gov (United States)

    ... Genetics of Skin Cancer Skin Cancer Screening Research Skin Cancer Prevention (PDQ®)–Patient Version What is prevention? ... prevent cancer are being studied. General Information About Skin Cancer Key Points Skin cancer is a disease ...

  4. Skin Cancer Screening

    Science.gov (United States)

    ... Genetics of Skin Cancer Skin Cancer Screening Research Skin Cancer Screening (PDQ®)–Patient Version What is screening? ... These are called diagnostic tests . General Information About Skin Cancer Key Points Skin cancer is a disease ...

  5. Dry Skin (Xerosis)

    Science.gov (United States)

    ... Kids’ zone Video library Find a dermatologist Dry skin Overview Dry, ashy skin: People who had atopic ... often have very dry skin as adults. Dry skin: Overview Also called xerosis Dry skin is common. ...

  6. Reduced dermis thickness and AGE accumulation in diabetic abdominal skin.

    Science.gov (United States)

    Niu, Yiwen; Cao, Xiaozan; Song, Fei; Xie, Ting; Ji, Xiaoyun; Miao, Mingyuan; Dong, Jiaoyun; Tian, Ming; Lin, Yuan; Lu, Shuliang

    2012-09-01

    Dermatological problems in diabetes might play an important role in the spontaneous ulcers and impaired wound healing that are seen in diabetic patients. Investigation of the cause of diabetic skin disorders is critical for identifying effective treatment. The abdominal full-thickness skin tissues of 33 patients (14 nondiabetic and 19 diabetic) were analyzed. The cell viability and malondialdehyde (MDA) production of fibroblasts were measured after advanced glycosylation end product (AGE)-bovine serum albumin (BSA) exposure. Cutaneous histological observation showed reduced thickness of the diabetic abdominal dermis with morphological characteristics of obscured multilayer epithelium and shortened, thinned, and disorganized collagen fibrils with focal chronic inflammatory cell infiltration when compared with controls of the same age. Accumulation of AGEs in diabetic skin was prominent. Less hydroxyproline, higher myeloperoxidase activity, and increased MDA content were detected in diabetic skin. In vitro, the time- and dose-dependent inhibitory effects of AGE-BSA on fibroblast viability as well as the fact that AGE-BSA could promote MDA production of fibroblasts were shown. It is shown that the accumulation of AGEs in diabetic skin tissue induces an oxidative damage of fibroblasts and acts as an important contributor to the thinner diabetic abdominal dermis. The authors believe that diabetic cutaneous properties at baseline may increase the susceptibility to injury, and diabetic wounds possess atypical origin in the repair process.

  7. Anti-wrinkle effects of Sargassum muticum ethyl acetate fraction on ultraviolet B-irradiated hairless mouse skin and mechanistic evaluation in the human HaCaT keratinocyte cell line.

    Science.gov (United States)

    Song, Jae Hyoung; Piao, Mei Jing; Han, Xia; Kang, Kyoung Ah; Kang, Hee Kyoung; Yoon, Weon Jong; Ko, Mi Hee; Lee, Nam Ho; Lee, Mi Young; Chae, Sungwook; Hyun, Jin Won

    2016-10-01

    The present study investigated the photoprotective properties of the ethyl acetate fraction of Sargassum muticum (SME) against ultraviolet B (UVB)‑induced skin damage and photoaging in a mouse model. HR‑1 strain hairless male mice were divided into three groups: An untreated control group, a UVB‑irradiated vehicle group and a UVB‑irradiated SME group. The UVB‑irradiated mice in the SME group were orally administered with SME (100 mg/kg body weight in 0.1 ml water per day) and then exposed to radiation at a dose of 60‑120 mJ/cm2. Wrinkle formation and skin damage were evaluated by analysis of skin replicas, epidermal thickness and collagen fiber integrity in the dermal connective tissue. The mechanism underlying the action of SME was also investigated in the human HaCaT keratinocyte cell line following exposure of the cells to UVB at a dose of 30 mJ/cm2. The protein expression levels and activity of matrix metalloproteinase‑1 (MMP‑1), and the binding of activator protein‑1 (AP‑1) to the MMP‑1 promoter were assessed in the HaCaT cells using western blot analysis, an MMP‑1 fluorescent assay and a chromatin immune‑precipitation assay, respectively. The results showed that the mean length and depth of the wrinkles in the UVB‑exposed hairless mice were significantly improved by oral administration of SME, which also prevented the increase in epidermal thickness triggered by UVB irradiation. Furthermore, a marked increase in collagen bundle formation was observed in the UVB‑treated mice with SME administration. SME pretreatment also significantly inhibited the UVB‑induced upregulation in the expression and activity of MMP‑1 in the cultured HaCaT keratinocytes, and the UVB‑enhanced association of AP‑1 with the MMP‑1 promoter. These results suggested that SME may be useful as an anti-photoaging resource for the skin.

  8. Fibroblasts from long-lived Snell dwarf mice are resistant to oxygen-induced in vitro growth arrest

    DEFF Research Database (Denmark)

    Maynard, Scott P; Miller, Richard A

    2006-01-01

    Snell dwarf mice live longer than controls, and show lower age-adjusted rates of lethal neoplastic diseases. Fibroblast cells from adult dwarf mice are resistant to the lethal effects of oxidative and nonoxidative stresses, including the carcinogen methyl methanesulfonate. We now report that dwarf...... in skin fibroblasts by the hormonal milieu of the Snell dwarf lead to resistance to multiple forms of injury, including the oxidative damage that contributes to growth arrest in vitro and neoplasia in intact mice....

  9. Simultaneous Reprogramming and Gene Correction of Patient Fibroblasts

    Directory of Open Access Journals (Sweden)

    Sara E. Howden

    2015-12-01

    Full Text Available The derivation of genetically modified induced pluripotent stem (iPS cells typically involves multiple steps, requiring lengthy cell culture periods, drug selection, and several clonal events. We report the generation of gene-targeted iPS cell lines following a single electroporation of patient-specific fibroblasts using episomal-based reprogramming vectors and the Cas9/CRISPR system. Simultaneous reprogramming and gene targeting was tested and achieved in two independent fibroblast lines with targeting efficiencies of up to 8% of the total iPS cell population. We have successfully targeted the DNMT3B and OCT4 genes with a fluorescent reporter and corrected the disease-causing mutation in both patient fibroblast lines: one derived from an adult with retinitis pigmentosa, the other from an infant with severe combined immunodeficiency. This procedure allows the generation of gene-targeted iPS cell lines with only a single clonal event in as little as 2 weeks and without the need for drug selection, thereby facilitating “seamless” single base-pair changes.

  10. Applications of human skin in vitro

    OpenAIRE

    Lönnqvist, Susanna

    2016-01-01

    Chronic wounds are a substantial problem in today’s health care and place significant strains on the patient. Successful modelling of the wound healing process is pivotal for the advancement of wound treatment research. Wound healing is a dynamic and multifactorial process involving all constituents of the skin. The progression from haemostasis and inflammation to proliferation of epidermal  keratinocytes and dermal fibroblasts, and final scar maturation can be halted and result in a chronic ...

  11. STAT6-dependent collagen synthesis in human fibroblasts is induced by bovine milk

    OpenAIRE

    Stefan Kippenberger; Nadja Zöller; Johannes Kleemann; Jutta Müller; Roland Kaufmann; Matthias Hofmann; August Bernd; Markus Meissner; Eva Valesky

    2015-01-01

    Since the domestication of the urus, 10.000 years ago, mankind utilizes bovine milk for different purposes. Besides usage as a nutrient also the external application of milk on skin has a long tradition going back to at least the ancient Aegypt with Cleopatra VII as a great exponent. In order to test whether milk has impact on skin physiology, cultures of human skin fibroblasts were exposed to commercial bovine milk. Our data show significant induction of proliferation by milk (max. 2,3-fold,...

  12. Comparison of epidermal keratinocytes and dermal fibroblasts as potential target cells for somatic gene therapy of phenylketonuria

    DEFF Research Database (Denmark)

    Christensen, Rikke; Güttler, Flemming; Jensen, Thomas G

    2002-01-01

    gene therapy. We have previously shown that overexpression of PAH and GTP-CH in primary human keratinocytes leads to high levels of phenylalanine clearance without BH(4) supplementation [Gene Ther. 7 (2000) 1971]. Here, we investigate the capacity of fibroblasts, another cell type from the skin......, to metabolize phenylalanine. After retroviral gene transfer of PAH and GTP-CH both normal and PKU patient fibroblasts were able to metabolize phenylalanine, however, in lower amounts compared to genetically modified keratinocytes. Further comparative analyses between keratinocytes and fibroblasts revealed...

  13. Aging and senescence of skin cells in culture

    DEFF Research Database (Denmark)

    Rattan, Suresh

    2015-01-01

    aging in vitro are dermal fibroblasts, epidermal keratinocytes, and melanocytes. Serial subcultivation of normal diploid skin cells can be performed only a limited number of times, and the emerging senescent phenotype can be categorized into structural, physiological, biochemical, and molecular......Studying age-related changes in the physiology, biochemistry, and molecular biology of isolated skin cell populations in culture has greatly expanded the understanding of the fundamental aspects of skin aging. The three main cell types that have been studied extensively with respect to cellular...... phenotypes, which can be used as biomarkers of cellular aging in vitro. The rate and phenotype of aging are different in different cell types. There are both common features and specific features of aging of skin fibroblasts, keratinocytes, melanocytes, and other cell types. A progressive accumulation...

  14. Asiaticoside enhances normal human skin cell migration, attachment and growth in vitro wound healing model.

    Science.gov (United States)

    Lee, Jeong-Hyun; Kim, Hye-Lee; Lee, Mi Hee; You, Kyung Eun; Kwon, Byeong-Ju; Seo, Hyok Jin; Park, Jong-Chul

    2012-10-15

    Wound healing proceeds through a complex collaborative process involving many types of cells. Keratinocytes and fibroblasts of epidermal and dermal layers of the skin play prominent roles in this process. Asiaticoside, an active component of Centella asiatica, is known for beneficial effects on keloid and hypertrophic scar. However, the effects of this compound on normal human skin cells are not well known. Using in vitro systems, we observed the effects of asiaticoside on normal human skin cell behaviors related to healing. In a wound closure seeding model, asiaticoside increased migration rates of skin cells. By observing the numbers of cells attached and the area occupied by the cells, we concluded that asiaticoside also enhanced the initial skin cell adhesion. In cell proliferation assays, asiaticoside induced an increase in the number of normal human dermal fibroblasts. In conclusion, asiaticoside promotes skin cell behaviors involved in wound healing; and as a bioactive component of an artificial skin, may have therapeutic value.

  15. Study of mast cell count in skin tags

    Directory of Open Access Journals (Sweden)

    Zaher Hesham

    2007-01-01

    Full Text Available Background: Skin tags or acrochordons are common tumors of middle-aged and elderly subjects. They consist of loose fibrous tissue and occur mainly on the neck and major flexures as small, soft, pedunculated protrusions. Objectives: The aim was to compare the mast cells count in skin tags to adjacent normal skin in diabetic and nondiabetic participants in an attempt to elucidate the possible role of mast cells in the pathogenesis of skin tags. Participants and Methods: Thirty participants with skin tags were divided into group I (15 nondiabetic participants and group II (15 diabetic participants. Three biopsies were obtained from each participant: a large skin tag, a small skin tag and adjacent normal skin. Mast cell count from all the obtained sections was carried out, and the mast cell density was expressed as the average mast cell count/high power field (HPF. Results: A statistically significant increase in mast cells count in skin tags in comparison to normal skin was detected in group I and group II. There was no statistically significant difference between mast cell counts in skin tags of both the groups. Conclusion: Both the mast cell mediators and hyperinsulinemia are capable of inducing fibroblast proliferation and epidermal hyperplasia that are the main pathologic abnormalities seen in all types of skin tags. However, the presence of mast cells in all examined skin tags regardless of diabetes and obesity may point to the possible crucial role of mast cells in the etiogenesis of skin tags through its interaction with fibroblasts and keratinocytes.

  16. Protective effect of bioactive peptides from taihe black-bone silky fowls on human skin fibroblasts injured by 5-fluorouracil%泰和乌骨鸡活性肽对5-氟尿嘧啶诱导HSF细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    田颖刚; 王春艳; 黄宇玫; 廖春艳; 张盼; 朱胜; 乔娟娟

    2012-01-01

    目的:用单细胞凝胶电泳技术(SCGE)研究泰和乌骨鸡活性肽对5-氟尿嘧啶(5-FU)损伤细胞DNA的保护作用。方法:传代培养人皮肤成纤维细胞(HSF),随机分为正常对照组,5-FU组,5-FU+泰和乌骨鸡活性肽组。应用MTT法检测细胞存活率,单细胞凝胶电泳法(SCGE)检测5-FU引起HSF细胞DNA损伤程度,羟脯氨酸试剂盒检测细胞培养液中羟脯氨酸含量。结果:与正常对照组比较:5-FU组HSF细胞DNA的损伤程度较严重,且尾长、尾距、Olive尾距和尾部DNA百分含量显著增加(P〈0.001),细胞培养液中羟脯氨酸含量显著性减少(P〈0.05)。与5-FU组比较,泰和乌骨鸡活性肽组细胞的尾长、尾距、Olive尾距和尾部DNA百分含量均显著性减少(P〈0.001),而细胞培养液上清中羟脯氨酸含量升高(P〈0.01)。结论:泰和乌骨鸡活性肽对HSF细胞DNA损伤具有保护作用,泰和乌骨鸡活性肽可以剂量依赖性地降低5-FU诱导的HSF细胞DNA的断裂损伤。%Objective : A DNA injury model was established by using 5- fluorouracil ( 5- FU ) in this study. Methods : A series of experiments ( MTT assay, single cell gel electrophoresis ( SCGE), and hydroxyproline assay kits) were designed to investigate the effect of bioactive peptides from Taihe Black-Bone Silky FowI(TBSF)on Human Skin Fibroblasts (HSF) against 5- FU- induced DNA damage.Cultured HSF were randomly divided into three groups : the control group,5-fluorouracil group and 5-fluorouracil + bioactive peptides from TBSF.Cell viability was measured by MTT assay, DNA damage was detected by SCGE assay, and hydroxyproline levels in HSF culture medium were measured by hydroxyproline assay kits. Results: compared with the control group,5-FU group exacerbated the DNA damage,in which the tail length,the tail moment,the Olive tail moment,and the tail DNA content significantly increased ( p 〈 0.001 ), whereas the

  17. Co-cultivation of pancreatic cancer cells with orthotopic tumor-derived fibroblasts: fibroblasts stimulate tumor cell invasion via HGF secretion whereas cancer cells exert a minor regulative effect on fibroblasts HGF production.

    Science.gov (United States)

    Qian, Li-Wu; Mizumoto, Kazuhiro; Maehara, Naoki; Ohuchida, Kenoki; Inadome, Naoki; Saimura, Michiyo; Nagai, Eishi; Matsumoto, Kunio; Nakamura, Toshikazu; Tanaka, Masao

    2003-02-10

    The intensive stromal reaction is one of characteristics of pancreatic exocrine carcinoma. The mutual interaction between pancreatic cancer cells and orthotopic tumor-derived fibroblasts have not been clarified yet. In this study, we sought to elucidate the mechanism underlying the tumor-stromal interaction with an in vitro coculture experimental system. Considerable strong c-Met expression was detected in seven out ten lines of human pancreatic carcinoma cells, as determined by Western blotting. For hepatocyte growth factor (HGF)-production, however, none or only trace amounts of HGF could be detected in those ten cell lines. Of the two lots of tumor-derived fibroblasts obtained from two pancreatic cancer patients, the fibroblasts capable to produce HGF could initiate an apparent invasion-stimulating response in strong c-Met-expressed Suit-2 and Panc-1 cells but not in faint expressed Mia PaCa-2 and BxPC-3 cells. A specialized HGF antagonist, NK4 would effectively inhibit the fibroblast-mediated invasive growth, thus proving the key role of the paracrine-fashioned HGF/c-Met pathway in the tumor-stromal interaction. On the other hand, the regulative action of cancer cells on HGF expression of fibroblasts was also investigated using direct or indirect coculture systems. For the fibroblasts that originally did not produce HGF, cancer cells failed to show any HGF-inductive effect. For the HGF-producing fibroblasts, despite of somewhat upregulation or downregulation in fibroblast HGF expression, the feedback regulation by studied pancreatic cancer cells in both coculture modes were relatively limited. This in vitro study sketched out the interaction between cancerous and stromal compartments with an emphasis on HGF/c-Met signal pathway, thus possibly helping to unveil the more complicated mutual modulation in vivo between pancreatic cancer and host mesenchymal tissues.

  18. A new dermocosmetic containing retinaldehyde, delta-tocopherol glucoside and glycylglycine oleamide for managing naturally aged skin: results from in vitro to clinical studies

    Directory of Open Access Journals (Sweden)

    Rouvrais C

    2017-02-01

    Full Text Available Céline Rouvrais,1,* Daniel Bacqueville,2,* Patrick Bogdanowicz,2,* Marie-José Haure,2 Laure Duprat,2 Christine Coutanceau,3 Nathalie Castex-Rizzi,2 Hélène Duplan,2 Valérie Mengeaud,1 Sandrine Bessou-Touya2 1Clinical Skin Research Center, 2Department of Pharmacology, Pierre Fabre Dermo-Cosmétique, Toulouse, 3Laboratoire Dermatologique Avène, Lavaur, France *These authors contributed equally to this work Introduction: Natural aging of skin tissues, the addition of the cumulative action of the time and radiation exposure result in skin atrophy, wrinkles and degeneration of the extracellular matrix (ECM. The aim of the study was to investigate the beneficial effect of a combination containing retinaldehyde (RAL, delta-tocopherol glucoside (delta-TC and glycylglycine oleamide (GGO and of a dermocosmetic containing the combination. Materials and methods: The protective effect of the combination was assessed through in vitro gene expression of ultraviolet (UV-irradiated fibroblasts. A skin aging assay using UV light on ex vivo skin samples and a clinical study conducted in 36 women aged from 35 to 55 years with a minimum of level 4 to a maximum of level 6 on the crow’s feet photoscale assessed the antiaging effect of the dermocosmetic. Results: When added to UV-irradiated fibroblasts, the combination substantially improved the ECM in activating the elastin fiber production (fibrillin 2, fibulin 1 and 5 and lysyl ­oxidase-like 2 as well as that of proteins involved in the cellular ECM interactions (integrin β1, paxillin and actin a2. An ex vivo photodamaged human skin model showed that the dermocosmetic formulation containing the combination of the active ingredients protected the elastic network against UV-induced alterations including both elastin and fibrillin-rich fibers in the dermis. A daily application of the dermocosmetic for 2 months on naturally aged skin resulted in a statistically significant improvement (p<0.05 of visible

  19. Fibroblastic osteosarcoma of the mandible

    Energy Technology Data Exchange (ETDEWEB)

    Daffner, Richard H. [Department of Diagnostic Radiology, Allegheny General Hospital, Pittsburgh, PA (United States); Fox, Karl R. [Department of Laboratory Medicine, Allegheny General Hospital, Pittsburgh, PA (United States); Galey, Kent R. [Department of Surgery, Division of Oral and Maxillofacial Surgery, Allegheny General Hospital, Pittsburgh, PA (United States)

    2002-02-01

    Osteosarcoma of the mandible is a rare lesion. We report the case of a 16-year-old male who developed a fibroblastic osteosarcoma at the site of a wisdom tooth extraction. The lesion followed an aggressive course of recurrence and diffuse disseminated osteosarcomatosis. We speculate on the causal factors that resulted in this rapid course. (orig.)

  20. Construction of fat-1 adipose tissue specific expression vector and production of goat transgenic fibroblast cell line%fat-1基因脂肪组织特异性表达载体的构建及其山羊转基因细胞系的建立

    Institute of Scientific and Technical Information of China (English)

    陈建文; 刘星; 桂涛; 李运生; 章孝荣; 张瑾; 张运海

    2012-01-01

    The aim of this study is to construct a marker removable, fat-1 adipose tissue specific expression vector and produce the transgenic goat fibroblast cell line for nuclear transfer. Firstly, the fat-1 gene was syn-thezised and a fat-1 adipose tissue specific expression vector was constructed. Secondly, the adipose tissue specific expression cassette was subcloned into a marker removable backbone vector (MCS-3s-LoxP-RFP) to construct a fat-1 marker removable adipose tissue specific expression vector driven by mouse Fabp4 promoter. Fi-nally, the goat fetal fibroblasts was transfected with the vector by Lipofectmine 2000 and selected in medium with G418 for two weeks, and then G418 resistant transfectants were identified by PCR. The results showed that the fat-1 marker removable adipose tissue specific expression vector was successfully constructed and the transgenic goat fibroblast cell lines were well established. It would pave the way for obtaining the marker-free fat-1 transgenic goat by SCNT.%旨在构建一种筛选标记可全部去除的脂肪组织特异性表达fat-1基因的载体,将其转染山羊胎儿成纤维细胞,筛选出稳定整合fat-1基因的转基因细胞系.首先将人工合成的fat-1基因连接至L28-Wnt10b载体(1种带有小鼠脂肪组织特异性启动子Fabp4的载体)上,构建成fat-1基因脂肪组织特异性表达载体L28-fat1;同时经多次克隆构建成1种筛选标记可全部去除的骨架载体MCS-3s-LoxP-RFP;然后,利用Hind Ⅲ和Not 1对上述2种载体进行双酶切,接着进行连接,构建出筛选标记可全部去除的脂肪组织特异性表达fat-1基因的表达载体.采用脂质体介导的方法转染山羊胎儿成纤维细胞,通过G418筛选转基因细胞.酶切鉴定及PCR检测结果表明,成功构建了3s-LoxP-RFP-FABP4-fat1表达载体,并首次获得了脂肪组织特异性表达fat-1基因的山羊胎儿成纤维转基因细胞系,为将来通过体细胞核移植创

  1. RAGE and TGF-β1 Cross-Talk Regulate Extracellular Matrix Turnover and Cytokine Synthesis in AGEs Exposed Fibroblast Cells.

    Directory of Open Access Journals (Sweden)

    Andreea Iren Serban

    Full Text Available AGEs accumulation in the skin affects extracellular matrix (ECM turnover and triggers diabetes associated skin conditions and accelerated skin aging. The receptor of AGEs (RAGE has an essential contribution to cellular dysfunction driven by chronic inflammatory responses while TGF-β1 is critical in both dermal homeostasis and inflammation. We investigated the contribution of RAGE and TGF-β1 to the modulation of inflammatory response and ECM turnover in AGEs milieu, using a normal fibroblast cell line. RAGE, TGF-β1, collagen I and III gene and protein expression were upregulated after exposure to AGEs-BSA, and MMP-2 was activated. AGEs-RAGE was pivotal in NF-κB dependent collagen I expression and joined with TGF-β1 to stimulate collagen III expression, probably via ERK1/2 signaling. AGEs-RAGE axis induced upregulation of TGF-β1, TNF-α and IL-8 cytokines. TNF-α and IL-8 were subjected to TGF-β1 negative regulation. RAGE's proinflammatory signaling also antagonized AGEs-TGF-β1 induced fibroblast contraction, suggesting the existence of an inhibitory cross-talk mechanism between TGF-β1 and RAGE signaling. RAGE and TGF-β1 stimulated anti-inflammatory cytokines IL-2 and IL-4 expression. GM-CSF and IL-6 expression appeared to be dependent only on TGF-β1 signaling. Our data also indicated that IFN-γ upregulated in AGEs-BSA milieu in a RAGE and TGF-β1 independent mechanism. Our findings raise the possibility that RAGE and TGF-β1 are both involved in fibrosis development in a complex cross-talk mechanism, while also acting on their own individual targets. This study contributes to the understanding of impaired wound healing associated with diabetes complications.

  2. How to Create an Anti-Aging Skin Care Plan

    Science.gov (United States)

    ... for public Dermatology World Dialogues in Dermatology JAAD Mohs AUC MyDermPath+ Psoriasis Patient education resources Practice Management ... lines and wrinkles, and leathery skin. Eat healthy foods: A healthy diet promotes healthy skin. Make sure ...

  3. Skin Cancer

    Science.gov (United States)

    ... you ever get a bad sunburn that caused blisters when you were a child?Does your skin ... Cancer InstituteMelanoma International FoundationAmerican Cancer Society ResourcesDiagnosis and Management of Malignant Melanoma by BG Goldstein, AO Goldstein( ...

  4. Polycistronic lentivirus induced pluripotent stem cells from skin biopsies after long term storage, blood outgrowth endothelial cells and cells from milk teeth.

    Science.gov (United States)

    Dambrot, C; van de Pas, S; van Zijl, L; Brändl, B; Wang, J W; Schalij, M J; Hoeben, R C; Atsma, D E; Mikkers, H M; Mummery, C L; Freund, C

    2013-02-01

    The generation of human induced pluripotent stem cells (hiPSCs) requires the collection of donor tissue, but clinical circumstances in which the interests of patients have highest priority may compromise the quality and availability of cells that are eventually used for reprogramming. Here we compared (i) skin biopsies stored in standard physiological salt solution for up to two weeks (ii) blood outgrowth endothelial cells (BOECs) isolated from fresh peripheral blood and (iii) children's milk teeth lost during normal replacement for their ability to form somatic cell cultures suitable for reprogramming to hiPSCs. We derived all hiPSC lines using the same reprogramming method (a conditional (FLPe) polycistronic lentivirus) and under similar conditions (same batch of virus, fetal calf serum and feeder cells). Skin fibroblasts could be reprogrammed robustly even after long-term biopsy storage. Generation of hiPSCs from juvenile dental pulp cells gave similar high efficiencies, but that of BOECs was lower. In terms of invasiveness of biopsy sampling, biopsy storage and reprogramming efficiencies skin fibroblasts appeared best for the generation of hiPSCs, but where non-invasive procedures are required (e.g., for children and minors) dental pulp cells from milk teeth represent a valuable alternative.

  5. The Effects of Brazilian Green Propolis against Excessive Light-Induced Cell Damage in Retina and Fibroblast Cells

    Directory of Open Access Journals (Sweden)

    Hiromi Murase

    2013-01-01

    Full Text Available Background. We investigated the effects of Brazilian green propolis and its constituents against white light- or UVA-induced cell damage in mouse retinal cone-cell line 661W or human skin-derived fibroblast cells (NB1-RGB. Methods. Cell damage was induced by 3,000lx white light for 24 h or 4/10 J/cm2 UVA exposure. Cell viability was assessed by Hoechst33342 and propidium iodide staining or by tetrazolium salt (WST-8 cell viability assay. The radical scavenging activity of propolis induced by UVA irradiation in NB1-RGB cells was measured using a reactive-oxygen-species- (ROS- sensitive probe CM-H2DCFDA. Moreover, the effects of propolis on the UVA-induced activation of p38 and extracellular signal-regulated kinase (ERK were examined by immunoblotting. Results. Treatment with propolis and two dicaffeoylquinic acids significantly inhibited the decrease in cell viability induced by white light in 661W. Propolis and its constituents inhibited the decrease in cell viability induced by UVA in NB1-RGB. Moreover, propolis suppressed the intracellular ROS production by UVA irradiation. Propolis also inhibited the levels of phosphorylated-p38 and ERK by UVA irradiation. Conclusion. Brazilian green propolis may become a major therapeutic candidate for the treatment of AMD and skin damage induced by UV irradiation.

  6. Effect of periodontal dressings on human gingiva fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Eber, R.M.; Shuler, C.F.; Buchanan, W.; Beck, F.M.; Horton, J.E. (Ohio State Univ. College of Dentistry, Columbus (USA))

    1989-08-01

    In vitro cytotoxicity studies of periodontal dressings have not generally produced a result consistent with in vivo observations. These prior in vitro studies have not used human intraoral cell lines. We tested the effects of two eugenol containing and two non-eugenol periodontal dressings on cultured human gingival fibroblasts (HGF) (ATCC No. 1292). Replicate HGF cultures grown in microtiter plates were exposed to stock, 1:4 and 1:16 dilutions of extracts made from each of the four periodontal dressings. The HGF cultures were pulse labelled with tritiated thymidine (3HTdR) after 24, 48, and 72 hours. Incorporations of the labelled thymidine were measured using liquid scintillation counting and expressed as counts per minute. The results showed that undiluted extracts from all four periodontal dressings totally inhibited 3HTdR uptake (P less than 0.05). The 1:4 dilution of eugenol dressings inhibited 3HTdR uptake significantly more than non-eugenol dressings (P less than 0.05). Interestingly, at 72 hours the 1:16 dilution of the non-eugenol dressings caused significantly increased 3HTdR uptake which was not observed with the eugenol dressings. The present results suggest that the use of a human fibroblastic cell line for testing the effects of periodontal dressings may provide information about the relative biological effects of these dressings. Using this cell line, we have found that eugenol dressings inhibit fibroblast proliferation to a greater extent than non-eugenol dressings.

  7. Human Dermal Stem/Progenitor Cell-Derived Conditioned Medium Improves Senescent Human Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Ji-Yong Jung

    2015-08-01

    Full Text Available Adult skin stem cells are recognized as potential therapeutics to rejuvenate aged skin. We previously demonstrated that human dermal stem/progenitor cells (hDSPCs with multipotent capacity could be enriched from human dermal fibroblasts using collagen type IV. However, the effects of hDSPCs on cellular senescence remain to be elucidated. In the present study, we investigated whether conditioned medium (CM collected from hDSPC cultures (hDSPC-CM exhibits beneficial effects on senescent fibroblasts. We found that hDSPC-CM promoted proliferation and decreased the expression level of senescence-associated β-galactosidase in senescent fibroblasts. In addition, p53 phosphorylation and p21 expression were significantly reduced in senescent fibroblasts treated with hDSPC-CM. hDSPC-CM restored the expression levels of collagen type I, collagen type III, and tissue inhibitor of metalloproteinase, and antagonized the increase of matrix metalloproteinase 1 expression. Finally, we demonstrated that hDSPC-CM significantly reduced reactive oxygen species levels by specifically up-regulating the expression level of superoxide dismutase 2. Taken together, these data suggest that hDSPC-CM can be applied as a potential therapeutic agent for improving human aged skin.

  8. Squamous cell skin cancer

    Science.gov (United States)

    ... that reflect light more, such as water, sand, concrete, and areas that are painted white. The higher ... - skin - squamous cell; Skin cancer - squamous cell; Nonmelanoma skin cancer - squamous ...

  9. Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound Efeitos do fator de crescimento de fibroblastos básico e do seu anti-fator na cicatrização e maturação do colágeno de feridas infectadas de pele

    Directory of Open Access Journals (Sweden)

    Antonio Medeiros Dantas Filho

    2007-01-01

    Full Text Available PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B. Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm², 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30 the wounds were contaminated with multibacterial standard solution, and in group B(n=30 the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis and F2 (for collagen study. The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering pOBJETIVO: Avaliar os efeitos do fator de crescimento de fibroblastos básico (FCFâ e do anti-FCFâ na cicatrização e maturação do colágeno em feridas infectadas na pele de ratos. MÉTODOS: Um estudo experimental foi realizado em 60 ratos Wistar, divididos em dois grupos (A e B. Cada grupo foi divididos em 03 subgrupos A1,B1; A2,B2 e A3,B3. Após anestesia com pentobarbital sódico intraperitoneal, foram feitas duas feridas abertas de 1cm² na pele no dorso distando 4cm uma da outra. Essas feridas foram

  10. Analysis of a Mouse Skin Model of Tuberous Sclerosis Complex

    Science.gov (United States)

    Guo, Yanan; Dreier, John R.; Cao, Juxiang; Du, Heng; Granter, Scott R.; Kwiatkowski, David J.

    2016-01-01

    Tuberous Sclerosis Complex (TSC) is an autosomal dominant tumor suppressor gene syndrome in which patients develop several types of tumors, including facial angiofibroma, subungual fibroma, Shagreen patch, angiomyolipomas, and lymphangioleiomyomatosis. It is due to inactivating mutations in TSC1 or TSC2. We sought to generate a mouse model of one or more of these tumor types by targeting deletion of the Tsc1 gene to fibroblasts using the Fsp-Cre allele. Mutant, Tsc1ccFsp-Cre+ mice survived a median of nearly a year, and developed tumors in multiple sites but did not develop angiomyolipoma or lymphangioleiomyomatosis. They did develop a prominent skin phenotype with marked thickening of the dermis with accumulation of mast cells, that was minimally responsive to systemic rapamycin therapy, and was quite different from the pathology seen in human TSC skin lesions. Recombination and loss of Tsc1 was demonstrated in skin fibroblasts in vivo and in cultured skin fibroblasts. Loss of Tsc1 in fibroblasts in mice does not lead to a model of angiomyolipoma or lymphangioleiomyomatosis. PMID:27907099

  11. Water-filtered near-infrared influences collagen synthesis of keloid-fibroblasts in contrast to normal foreskin fibroblasts.

    Science.gov (United States)

    Zöller, Nadja; König, Anke; Butting, Manuel; Kaufmann, Roland; Bernd, August; Valesky, Eva; Kippenberger, Stefan

    2016-10-01

    Hypertrophic scar development is associated to impaired wound healing, imbalanced fibroblast proliferation and extracellular matrix synthesis. Stigmatization, physical restrictions and high recurrence rates are only some aspects that illustrate the severe influence impaired wound healing can have on patients' life. The treatment of hypertrophic scars especially keloids is still a challenge. In recent years water-filtered near-infrared irradiation (wIRA) composed of near-infrared (NIR) and a thermal component is applied for an increasing penal of clinical purposes. It is described to beneficially influence e.g. wound healing. But discrimination between the thermal and the NIR dependent components of these effects has not been conclusively elucidated. Aim of our study was therefore to investigate the influence of the light fraction on the thermal impact of wIRA irradiation in dermal cells. We concentrated our analysis on morphological properties and collagen synthesis. Foreskin fibroblasts and the keloid fibroblast cell line KF111 were exposed to temperatures between 37°C and 46°C with or without additional irradiation with 360J/cm(2) NIR. Our results show that viability was not influenced by irradiation. Independent of the analysed fibroblast species temperature dependent occurrence of spheric cells could be observed. These morphological changes were clearly counteracted by additional light exposure. Convective heat reduced collagen type I synthesis in both cell species depending on the applied temperature. Co-treatment with NIR significantly reversed this effect in keloid fibroblast cultures treated at 46°C whereas no difference could be observed in the foreskin fibroblasts. The observed influence on collagen type I synthesis was associated to a temperature dependent TGF-β1 secretion reduction. Co-stimulation of keloid cultures with NIR at 46°C completely abolished the temperature dependent TGF-β1 secretion reduction. In foreskin fibroblast cultures co

  12. Normal human fibroblasts produce membrane-bound and soluble isoforms of FGFR-1.

    Science.gov (United States)

    Root, L L; Shipley, G D

    2000-02-01

    Fibroblast growth factors (FGFs) are polypeptide mitogens for a wide variety of cell types and are involved in other processes such as angiogenesis and cell differentiation. FGFs mediate their biological responses by activating high-affinity tyrosine kinase receptors. Currently, there are four human fibroblast growth factor receptor (FGFR) genes. To investigate the mechanisms by which alpha FGF and beta FGF may mediate mitogenic signal transduction in human skin-derived fibroblasts, we analyzed these cells for the presence of high-affinity FGFRs. We show that normal human dermal fibroblasts express a single high-affinity FGFR gene, FGFR-1. Cloning and sequencing of two distinct FGFR-1 cDNAs suggested that normal human dermal fibroblasts express a membrane-bound and a putatively secreted form of FGFR-1. We show that normal human dermal fibroblasts produce two FGFR-1 proteins, one of which exists in conditioned media. The mRNA for the putatively secreted form of FGFR-1 appears to be down-regulated by serum treatment of the cells.

  13. Pentadecapeptide BPC 157 Enhances the Growth Hormone Receptor Expression in Tendon Fibroblasts

    Directory of Open Access Journals (Sweden)

    Chung-Hsun Chang

    2014-11-01

    Full Text Available BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon.

  14. Deletion of the homeobox gene PRX-2 affects fetal but not adult fibroblast wound healing responses.

    Science.gov (United States)

    White, Philip; Thomas, David W; Fong, Steven; Stelnicki, Eric; Meijlink, Fritz; Largman, Corey; Stephens, Phil

    2003-01-01

    The phenotype of fibroblasts repopulating experimental wounds in vivo has been shown to influence both wound healing responses and clinical outcome. Recent studies have demonstrated that the human homeobox gene PRX-2 is strongly upregulated in fibroblasts within fetal, but not adult, mesenchymal tissues during healing. Differential homeobox gene expression by fibroblasts may therefore be important in mediating the scarless healing exhibited in early fetal wounds. RNase protection analysis demonstrated that murine Prx-2 expression was involved in fetal but not adult wound healing responses in vitro. Using fibroblasts established from homozygous mutant (Prx-2-/-) and wild-type (Prx-2+/+) murine skin tissues it was demonstrated that Prx-2 affected a number of fetal fibroblastic responses believed to be important in mediating scarless healing in vivo; namely cellular proliferation, extracellular matrix reorganization, and matrix metalloproteinase 2 and hyaluronic acid production. These data demonstrate how Prx-2 may contribute to the regulation of fetal, but not adult, fibroblasts and ultimately the wound healing phenotype. This study provides further evidence for the importance of homeobox transcription factors in the regulation of scarless wound healing. A further understanding of these processes will, it is hoped, enable the targeting of specific therapies in wound healing, both to effect scarless healing and to stimulate healing in chronic, nonhealing wounds such as venous leg ulcers.

  15. Radiation sensitivity of Merkel cell carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Leonard, J.H.; Ramsay, J.R.; Birrell, G.W. [Queensland Institute of Medical Research (Australia)] [and others

    1995-07-30

    Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after {gamma} irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to {gamma} irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution. 25 refs., 3 figs., 1 tab.

  16. Skin Cancer Foundation

    Science.gov (United States)

    ... Host a Fundraising Event | About Us | Store The Skin Cancer Foundation The Skin Cancer Foundation is the ... A "Sunscreen Gene"? Skin Cancer Facts & Statistics The Skin Cancer Foundation's Champions for Change Gala 2016 Learn ...

  17. Skin Pigmentation Disorders

    Science.gov (United States)

    Pigmentation means coloring. Skin pigmentation disorders affect the color of your skin. Your skin gets its color from a pigment called melanin. Special cells in the skin make melanin. When these cells become damaged or ...

  18. Annexin A1 N-terminal derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions.

    Directory of Open Access Journals (Sweden)

    Valentina Bizzarro

    Full Text Available Deficient wound healing in diabetic patients is very frequent, but the cellular and molecular causes are poorly defined. In this study, we have evaluated whether Annexin A1 derived peptide Ac2-26 stimulates fibroblast migration in high glucose conditions. Using normal human skin fibroblasts WS1 in low glucose (LG or high glucose (HG we observed the enrichment of Annexin A1 protein at cell movement structures like lamellipodial extrusions and interestingly, a significant decrease in levels of the protein in HG conditions. The analysis of the translocation of Annexin A1 to cell membrane showed lower levels of Annexin A1 in both membrane pool and supernatants of WS1 cells treated with HG. Wound-healing assays using cell line transfected with Annexin A1 siRNAs indicated a slowing down in migration speed of cells suggesting that Annexin A1 has a role in the migration of WS1 cells. In order to analyze the role of extracellular Annexin A1 in cell migration, we have performed wound-healing assays using Ac2-26 showing that peptide was able to increase fibroblast cell migration in HG conditions. Experiments on the mobilization of intracellular calcium and analysis of p-ERK expression confirmed the activity of the FPR1 following stimulation with the peptide Ac2-26. A wound-healing assay on WS1 cells in the presence of the FPR agonist fMLP, of the FPR antagonist CsH and in the presence of Ac2-26 indicated that Annexin A1 influences fibroblast cell migration under HG conditions acting through FPR receptors whose expression was slightly increased in HG. In conclusion, these data demonstrate that (i Annexin A1 is involved in migration of WS1 cells, through interaction with FPRs; (ii N- terminal peptide of Annexin A1 Ac2-26 is able to stimulate direct migration of WS1 cells in high glucose treatment possibly due to the increased receptor expression observed in hyperglycemia conditions.

  19. Skin Bleaching

    OpenAIRE

    Ahmed, Samira M.

    2015-01-01

    In this project, I aim to investigate the reasoning behind the practice of skin bleaching by analyzing the documentary ”Dark Girls”, to gain a better understanding of race and colorism issues. Also this project tries to see if there is a connection with history and if this has been a part of making the european beauty ideal determine the choices black’s make in regards to beauty.

  20. Single Exposure of Human Oral Mucosa Fibroblasts to Ultraviolet B Radiation Reduces Proliferation and Induces COX-2 Expression and Activation

    Directory of Open Access Journals (Sweden)

    Y Boza

    2010-12-01

    Full Text Available The lip vermillion constitutes a transition tissue, between oral mucosa and skin, where oral mucosal cells from epithelial and connective tissue compartments are exposed to ultraviolet (UV sunlight. Fibroblasts are abundant resident cells of the connective tissue which are key regulators of extracellular matrix composition, as well as, epithelial and endothelial cell function. UVB light, an inherent component of sunlight, causes several alterations in skin fibroblasts, including premature senescence and increased cyclooxygenase (COX-2 expression. To assess if UVB irradiation had similar effects on fibroblasts derived from human oral mucosa (HOM, primary cultures of HOM fibroblasts were irradiated with a single dose of 30 or 60 mJ/cm²of UVB light or sham-irradiated. Fibroblast proliferation was assessed from 3 to 48 hrs after UVB-irradiation utilizing [³H]-thymidine incorporation and MTT assays. In addition, COX-2 mRNA expression was detected by RT-PCR, and PGE2 production was assessed using enzyme immunoassay from 0.5 to 24 hrs after UVB-irradiation. The results showed a significant decrease in proliferation of UVB-irradiated HOM fibroblasts as compared to controls as measured by both [³H]-thymidine incorporation and MTT assays (p<0.001. HOM fibroblasts had increased COX-2 mRNA expression at 0.5 and 12 hrs after irradiation, and PGE2 production was elevated at 12 and 24 hrs post-irradiation as compared to controls (p<0.05. The results showed an inhibitory effect of a single dose of UVB irradiation on HOM fibroblast proliferation with an increase in COX-2 expression and activation. Therefore, photodamaged fibroblasts may play and important role in the pathogenesis of UV-induced lesions of the lip.

  1. The lysyl oxidase LOX is absent in basal and squamous cell carcinomas and its knockdown induces an invading phenotype in a skin equivalent model.

    Science.gov (United States)

    Bouez, Charbel; Reynaud, Caroline; Noblesse, Emmanuelle; Thépot, Amélie; Gleyzal, Claudine; Kanitakis, Jean; Perrier, Eric; Damour, Odile; Sommer, Pascal

    2006-03-01

    Lysyl oxidase initiates the enzymatic stage of collagen and elastin cross-linking. Among five isoforms comprising the lysyl oxidase family, LOX is the better studied. LOX is associated to an antitumor activity in ras-transformed fibroblasts, and its expression is down-regulated in many carcinomas. The aim of this work was to shed light on LOX functions within the epidermis by studying its expression in human basal and squamous cell carcinomas and analyzing the effect of its enzymatic activity inhibition and protein absence on human keratinocytes behavior in a skin equivalent. In both carcinomas, LOX expression by epidermal tumor cells was lacking, while it was up-regulated around invading tumor cells in association with the stromal reaction. Lysyl oxidase activity inhibition using beta-aminoproprionitrile in a skin equivalent model prepared with both primary human keratinocytes and HaCaT cell line affected keratin 10 and filaggrin expression and disorganized the collagen network and the basement membrane. In spite of all these changes, no invasion phenotype was observed. Modelization of the invasive phenotype was only noticed in the skin equivalent developed with LOX antisense HaCaT cell line, where the protein LOX is specifically absent. Our results clearly indicate that lysyl oxidase enzymatic activity is essential not only for the integrity maintenance of the dermis but also for the homeostasis of the epidermis. Moreover, LOX protein plays a role in the skin carcinomas and invasion but not through its enzymatic activity.

  2. Effects of Essential Oils and Polyunsaturated Fatty Acids on Canine Skin Equivalents: Skin Lipid Assessment and Morphological Evaluation

    Directory of Open Access Journals (Sweden)

    S. Cerrato

    2013-01-01

    Full Text Available A canine skin equivalent model has been validated for the assessment of a topical formulation effects. Skin equivalents were developed from freshly isolated cutaneous canine fibroblasts and keratinocytes, after enzymatic digestion of skin samples (n=8 from different breeds. Fibroblasts were embedded into a collagen type I matrix, and keratinocytes were seeded onto its surface at air-liquid interface. Skin equivalents were supplemented with essential oils and polyunsaturated fatty acid formulation or with vehicle. Skin equivalents were histopathologically and ultrastructurally studied, and the three main lipid groups (free fatty acids, cholesterol, and ceramides were analyzed. Results showed that the culture method developed resulted in significant improvements in cell retrieval and confluence. Treated samples presented a thicker epidermis with increased number of viable cell layers, a denser and compact stratum corneum, and a more continuous basal membrane. Regarding lipid profile, treated skin equivalents showed a significant increase in ceramide content (51.7±1.3 when compared to untreated (41.6 ± 1.4 samples. Ultrastructural study evidenced a compact and well-organized stratum corneum in both treated and control skin equivalents. In conclusion, cell viability and ceramides increase, after lipid supplementation, are especially relevant for the treatment of skin barrier disruptions occurring in canine atopic dermatitis.

  3. Inhibition of normal human lung fibroblast growth by beryllium.

    Science.gov (United States)

    Lehnert, N M; Gary, R K; Marrone, B L; Lehnert, B E

    2001-03-07

    Inhalation of particulate beryllium (Be) and its compounds causes chronic Be disease (CBD) in a relatively small subset ( approximately 1-6%) of exposed individuals. Hallmarks of this pulmonary disease include increases in several cell types, including lung fibroblasts, that contribute to the fibrotic component of the disorder. In this regard, enhancements in cell proliferation appear to play a fundamental role in CBD development and progression. Paradoxically, however, some existing evidence suggests that Be actually has antiproliferative effects. In order to gain further information about the effects of Be on cell growth, we: (1) assessed cell proliferation and cell cycle effects of low concentrations of Be in normal human diploid fibroblasts, and (2) investigated the molecular pathway(s) by which the cell cycle disturbing effects of Be may be mediated. Treatment of human lung and skin fibroblasts with Be added in the soluble form of BeSO(4) (0.1-100 microM) caused inhibitions of their growth in culture in a concentration-dependent manner. Such growth inhibition was found to persist, even after cells were further cultured in Be(2+)-free medium. Flow cytometric analyses of cellular DNA labeled with the DNA-binding fluorochrome DAPI revealed that Be causes a G(0)-G(1)/pre-S phase arrest. Western blot analyses indicated that the Be-induced G(0)-G(1)/pre-S phase arrest involves elevations in TP53 (p53) and the cyclin-dependent kinase inhibitor CDKN1A (p21(Waf-1,Cip1)). That Be at low concentrations inhibits the growth of normal human fibroblasts suggests the possibility of the existence of abnormal cell cycle inhibitory responses to Be in individuals who are sensitive to the metal and ultimately develop CBD.

  4. Melanocytes in the skin--comparative whole transcriptome analysis of main skin cell types.

    Science.gov (United States)

    Reemann, Paula; Reimann, Ene; Ilmjärv, Sten; Porosaar, Orm; Silm, Helgi; Jaks, Viljar; Vasar, Eero; Kingo, Külli; Kõks, Sulev

    2014-01-01

    Melanocytes possess several functions besides a role in pigment synthesis, but detailed characteristics of the cells are still unclear. We used whole transcriptome sequencing (RNA-Seq) to assess differential gene expression of cultivated normal human melanocytes with respect to keratinocytes, fibroblasts and whole skin. The present results reveal cultivated melanocytes as highly proliferative cells with possible stem cell-like properties. The enhanced readiness to regenerate makes melanocytes the most vulnerable cells in the skin and explains their high risk of developing into malignant melanoma.

  5. Cutaneous skin tag

    Science.gov (United States)

    Skin tag; Acrochordon; Fibroepithelial polyp ... have diabetes. They are thought to occur from skin rubbing against skin. ... The tag sticks out of the skin and may have a short, narrow stalk connecting it to the surface of the skin. Some skin tags are as long as ...

  6. Differentiation of human umbilical cord mesenchymal stem cells into dermal fibroblasts in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yanfu [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Chai, Jiake, E-mail: cjk304@126.com [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China); Sun, Tianjun; Li, Dongjie; Tao, Ran [Department of Burn and Plastic Surgery, Burns Institute, First Hospital Affiliated to General Hospital of PLA, Beijing (China)

    2011-10-07

    Highlights: {yields} Mesenchymal stem cells (MSCs) are potential seed cells for tissue-engineered skin. {yields} Tissue-derived umbilical cord MSCs (UCMSCs) can readily be isolated in vitro. {yields} We induce UCMSCs to differentiate into dermal fibroblasts via conditioned medium. {yields} Collagen type I and collagen type III mRNA level was higher in differentiated cells. {yields} UCMSCs-derived fibroblast-like cells strongly express fibroblast-specific protein. -- Abstract: Tissue-derived umbilical cord mesenchymal stem cells (UCMSCs) can be readily obtained, avoid ethical or moral constraints, and show excellent pluripotency and proliferation potential. UCMSCs are considered to be a promising source of stem cells in regenerative medicine. In this study, we collected newborn umbilical cord tissue under sterile conditions and isolated UCMSCs through a tissue attachment method. UCMSC cell surface markers were examined using flow cytometry. On the third passage, UCMSCs were induced to differentiate into dermal fibroblasts in conditioned induction media. The induction results were detected using immunofluorescence with a fibroblast-specific monoclonal antibody and real time PCR for type I and type III collagen. UCMSCs exhibited a fibroblast-like morphology and reached 90% confluency 14 to 18 days after primary culture. Cultured UCMSCs showed strong positive staining for CD73, CD29, CD44, CD105, and HLA-I, but not CD34, CD45, CD31, or HLA-DR. After differentiation, immunostaining for collagen type I, type III, fibroblast-specific protein, vimentin, and desmin were all strongly positive in induced cells, and staining was weak or negative in non-induced cells; total transcript production of collagen type I and collagen type III mRNA was higher in induced cells than in non-induced cells. These results demonstrate that UCMSCs can be induced to differentiate into fibroblasts with conditioned induction media and, in turn, could be used as seed cells for tissue

  7. Potent, selective inhibitors of fibroblast growth factor receptor define fibroblast growth factor dependence in preclinical cancer models.

    Science.gov (United States)

    Squires, Matthew; Ward, George; Saxty, Gordan; Berdini, Valerio; Cleasby, Anne; King, Peter; Angibaud, Patrick; Perera, Tim; Fazal, Lynsey; Ross, Douglas; Jones, Charlotte Griffiths; Madin, Andrew; Benning, Rajdeep K; Vickerstaffe, Emma; O'Brien, Alistair; Frederickson, Martyn; Reader, Michael; Hamlett, Christopher; Batey, Michael A; Rich, Sharna; Carr, Maria; Miller, Darcey; Feltell, Ruth; Thiru, Abarna; Bethell, Susanne; Devine, Lindsay A; Graham, Brent L; Pike, Andrew; Cosme, Jose; Lewis, Edward J; Freyne, Eddy; Lyons, John; Irving, Julie; Murray, Christopher; Newell, David R; Thompson, Neil T

    2011-09-01

    We describe here the identification and characterization of 2 novel inhibitors of the fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases. The compounds exhibit selective inhibition of FGFR over the closely related VEGFR2 receptor in cell lines and in vivo. The pharmacologic profile of these inhibitors was defined using a panel of human tumor cell lines characterized for specific mutations, amplifications, or translocations known to activate one of the four FGFR receptor isoforms. This pharmacology defines a profile for inhibitors that are likely to be of use in clinical settings in disease types where FGFR is shown to play an important role.

  8. A fibroblast-associated antigen: Characterization in fibroblasts and immunoreactivity in smooth muscle differentiated stromal cells

    DEFF Research Database (Denmark)

    Rønnov-Jessen, Lone; Celis, Julio E.; van Deurs, Bo

    1992-01-01

    Fibroblasts with smooth muscle differentiation are frequently derived from human breast tissue. Immunofluorescence cytochemistry of a fibroblast-associated antigen recognized by a monoclonal antibody (MAb), 1B10, was analyzed with a view to discriminating smooth muscle differentiated fibroblasts...... from vascular smooth muscle cells. The antigen was detected on the cell surface and in cathepsin D-positive and acridine orange-accumulating vesicular compartments of fibroblasts. Ultrastructurally, the antigen was revealed in coated pits and in endosomal and lysosomal structures. 1B10 recognized three...... immunoreactivity was specific to fibroblasts and smooth muscle differentiated fibroblasts within the context of vascular smooth muscle cells....

  9. Construction of synthetic dermis and skin based on a self-assembled peptide hydrogel scaffold.

    Science.gov (United States)

    Kao, Bunsho; Kadomatsu, Koichi; Hosaka, Yoshiaki

    2009-09-01

    Using biocompatible peptide hydrogel as a scaffold, we prepared three-dimensional synthetic skin that does not contain animal-derived materials or pathogens. The present study investigated preparation methods, proliferation, and functional expression of fibroblasts in the synthetic dermis and differentiation of keratinocytes in the epidermis. Synthetic dermis was prepared by mixing fibroblasts with peptide hydrogel, and synthetic skin was prepared by forming an epidermal layer using keratinocytes on the synthetic dermis. A fibroblast-rich foamy layer consisting of homogeneous peptide hydrogel subsequently formed in the synthetic dermis, with fibroblasts aggregating in clusters within the septum. The epidermis consisted of three to five keratinocyte layers. Immunohistochemical staining showed human type I collagen, indicating functional expression around fibroblasts in the synthetic dermis, keratinocyte differentiation in the epidermis, and expression of basement membrane proteins. The number of fibroblasts tended to increase until the second week and was maintained until the fourth week, but rapidly decreased in the fifth week. In the synthetic dermis medium, the human type I collagen concentration increased after the second week to the fifth week. These findings suggest that peptide hydrogel acts as a synthetic skin scaffold that offers a platform for the proliferation and functional expression of fibroblasts and keratinocytes.

  10. Use of autologous tissue engineered skin to treat porcine full-thickness skin defects

    Institute of Scientific and Technical Information of China (English)

    CAI Xia; CAO Yi-lin; CUI Lei; LIU Wei; GUAN Wen-xiang

    2005-01-01

    Objective: To explore a feasible method to repair full-thickness skin defects utilizing tissue engineered