郝丽; 李和平; 严厉
为克隆出与鹿茸生长发育相关基因的全长序列,采用SMART技术构建了东北梅花鹿鹿茸尖端组织的全长cDNA文库.用SV Total RNA Isolation System试剂盒提取总RNA,以逆转录酶PowerScriptTM 反转录合成第一链cDNA,然后通过LD-PCR合成并扩增ds cDNA.扩增产物经纯化、SfiⅠ酶切、过CHROMA SPIN-400柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB质粒载体中,最后用电转化法将重组质粒转化到E. coli DH5α内得到原始文库.经测定,构建的原始文库约含有2.56×10~6个重组子,插入片段多在0.5～2kb之间,平均插入片段长度约1.1kb,重组效率接近100%.结果表明,东北梅花鹿鹿茸尖端组织的全长cDNA文库已构建成功.%A study was conducted to construct full-length cDNA library from antler tip tissues of Sika Deer (Cenna nippon hortu-lonun) by SMART technique in order to clone new special genes for development of antler. The total RNA was extracted u-sing SV Total RNA Isolation System. Single-stranded cDNA was synthesized using PowerScripiTM reverse transcriptase,and double-stranded cDNA was synthesized and amplified by long-distance PCR. The PCR products were digested by pro-teinase K and purified. After digestion with Sfi I and size fractionation using CHROMA SPIN -400TM Columns, SMART cDNA was ligated to the Sfi I-digested, dephosphorylated pDNR-LIB vector, and the ligation mixture was transformed into E. call DH5a by electroporation. The primary cDNA library contained 2.56×10~6 independent clones with DNA inserts of 0.5～2. 0 kb, the average size of inserted cDNAs was 1.1 kb, and the recombination percentage was about 100%. Results showed that the full-length cDNA library from antler tip tissues of Sika Deer was successfully constructed.
宋光熠; 王晓颖; 段有金; 李开明
BACKGROUND: The incidence and progress of vascular dementia (VD)is closely related to the abnormal metabolism of oxygen-derived free radicals, whose metabolism is often the main index in pharmacodynamic experiments on dementia.OBJECTIVE: To establish the animal model of multiple cerebral infarction and determine the content of lipid peroxide and the activity of superoxide dismutase and glutathione peroxidase in the blood and cerebral tissues so as to analyze the effects of Cenma Yizhi Capsule on the oxidative status in vivo in VD rats.DESIGN: Randomized controlled single sample trial.SETTING: Department of Pharmacology, Institute of Basic Medicine of Liaoning Province.MATERIALS: This experiment was conducted in the Department of Pharmacology, Institute of Basic Medicine of Liaoning Province, between July 1997 and November 1998. Totally 96 healthy Wistar rats were recruited and randomized into 6 groups: normal control group, model control group ,positive drug control group, Cenma Yizhi Capsule high-dose group, Cenna Yizhi Capsule medium-dose group, and Cenma Yizhi Capsule low-dose group with 16 rats in each group. Cenma Yizhi Capsules, the compound of the total extract of ginseng, Tianma and other Chinese herbal medicine (each gram contained 2.7 g drug), were provided by Xiyuan Hospital of the Chinese Academy of Traditional Chinese Medicine.METHODS: The animal model of multiple cerebral infarction was established in all the groups except normal control group. Normal saline of 0.5 mL was gastrically perfused into the rats in Cenma Yizhi Capsule high-, medium- and low-dose groups; dihydroergotamine mesylate of 1mg/kg was gastrically perfused into the rats in positive drug control group. Rats in each group were given the drug 1 week after embolism, once a day for 6 consecutive weeks. The content of lipid peroxide was determined with fluorescence method, and the activity of superoxide dismutaseand glutathione peroxidase was determined with self-oxidation method of