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PCR扩增含有apoA-I基因的转录调控序列的启动子片断(376 bp),克隆入含有荧光素酶报告基因和新霉素抗性基因的pGL3B-neo载体中,构成受apoA-I启动子调控的重组报告基因质粒pGL3B-neo::apo,稳定转染人肝癌细胞株HepG2。通过细胞有限稀释法筛选稳定表达荧光素酶活性的细胞株SAPOA-I。经apoA-I转录调控因子PPARα激动剂的鉴定和细胞接种密度、溶剂使用浓度等条件的优化,成功建立了靶向apoA-I基因启动子转录活性的报告基因筛选体系。应用该体系对400多种降脂中药提取物进行筛选,泽泻、罗布麻叶、山楂等提取物显示出较好的上调活性,与文献报道采用动物模型研究的结果一致。
The promoter fragment (376 bp) containing the transcriptional regulatory sequence of the apoA-I gene was amplified by PCR and cloned into the pGL3B-neo vector containing the luciferase reporter gene and the neomycin resistance gene to construct a prokaryotic expression vector consisting of the apoA-I promoter Regulated recombinant plasmid pGL3B-neo :: apo, stably transfected into human hepatoma cell line HepG2. The cell line SAPOA-I stably expressing luciferase activity was screened by the cell limited dilution method. The screening system of reporter gene targeting the transcriptional activity of apoA-I promoter was successfully established through the identification of PPARα agonist of apoA-I transcription factor and optimization of cell seeding density and solvent concentration. More than 400 kinds of lipid-lowering traditional Chinese medicine extracts were screened by using this system. The extracts of Alisma orientalis, Apocynum venetum, hawthorn, etc. showed good up-regulation activity, which was consistent with the results of animal model studies reported in the literature.