摘要
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。
Amplified apolipoprotein A-Ⅰ （apoA-Ⅰ） promoter gene （ 376 bp） containing essential transcription regulatory elements from human genomic DNA was fused upstream to the luciferase reporter gene and neomycin resistance gene in the constructed pGL3B-neo vector. Then the reeombinant vector pGL3B-neo： ：apo regulated by apoA-Ⅰ promoter was introduced into HepG2 cell line. With limited cell dilution method in the presence of G418 monoclones were selected and finally the cell llne with stable highest luciferase activity was obtained as SAPOA-Ⅰ. At last ,this drug screening cell model in vitro based on apoA-Ⅰ promoter was established after identification by PPARα activators and optimization of cell density and DMSO final concentration. Then it was used to primarily screen nearly 400 samples extracted from traditional herbs with reported lipid-lowerlng effects, in which Alisma orientalis ,Folium apocyni veneti and Crataegus pinnatifida exhibited high relative lucifcrase activity,consistent with previous studies in animal models.
出处
《天然产物研究与开发》
CAS
CSCD
2007年第5期776-780,共5页
Natural Product Research and Development
