人高密度脂蛋白受体表达上调剂筛选模型的建立

Establishment of a Drug Screening Model for Identifying Up-regulator of Human High Density Lipoprotein Receptor

目的 建立靶向人高密度脂蛋白受体基因(CLA-1)调控序列的药物筛选模型,为筛选人高密度脂蛋白受体表达上调剂奠定基础。方法 PCR扩增CLA-1上游调控序列,构建重组报告基因质粒pGL3-CLAP,瞬时转染人肝癌细胞BEL-7402,通过检测荧光素酶报告基因表达水平的变化筛选人高密度脂蛋白受体表达上调剂。结果 建立了CLA-1表达上调剂筛选模型,pGL3-CLAP与阴性对照pGL3-Basic组间差异显著(P<0.001),变异系数(CV)不超过10％。对124个化合物和800个微生物次级代谢产物进行初筛和复筛,1个化合物和3个菌株发酵液为阳性。结论 该系统可有效地应用于人高密度脂蛋白受体表达上调剂的高通量筛选。

Objective To establish a new drug screening model based on transcriptional regulation of human high density lipoprotein (HDL) receptor gene CD36 and LIMP Ⅱ analogous-1 (CLA-1) for discovering up-regulator of this receptor. Methods The upstream regulatory sequence of CLA-1 was obtained by poly-merase chain reaction. A recombinant reporter plasmid pGL3-CLAP was constructed by inserting the regulatory sequence upstream of luciferase gene of pGL3-Basic. Human hepatoma cell line BEL-7402 was tr-ansfected with pGL3-CLAP. Samples were detected by testing luciferase activity of transfected BEL-7402 cells in microtiter wells. Results The drug screening model was established and optimized. Significant difference was present between pGL3-CLAP and pGL3-Basic transfected BEL-7402 cells (P < 0.001), and coefficient of variation was less than 10%. After primary and secondary screening, 1 compounds and 3 fermentation extracts had up-regulating activities. Conclusion This new drug screening model may be efficiently used to screen up - regulators of human HDL receptor expression, which might become lead compounds for new anti-atherosclerosis drugs.