建立一个基于报告基因的靶标筛药系统筛选上调ABCA1基因转录的中药提取物

ATP结合盒转运蛋白A1(ABCA1)具有催化外周细胞过量胆固醇和磷脂外向流出的功能。本实验将ABCA1启动子序列克隆到含有荧光素酶报告基因和新霉素抗性基因的pGL3B-neo载体中,并将得到的重组质粒pGL3B-neo/ABCA1转染入人肝癌细胞(hepG2)。通过稀释培养法最终得到含有重组质粒的稳定细胞系SABCA1。利用这个报告基因为基础的药物筛选系统,筛选了100多种中药提取物。通过荧光素酶活性测试,相对活性达到180%以上的有4种中药提取物,并最终挑选能明显激活ABCA1基因启动子的鬼箭羽水提组分进一步研究,得出半数有效浓度(EC50)为48.06 mg/L。此外,利用基于apoA1基因的药物筛选系统验证得出鬼箭羽水提组分也能促进apoA1基因的表达。

Establishment of a Reporter Gene-Based Drug Screening System for Identifying Herbal up-Regulators of ATP-Binding Cassette Transporter A1

ABCA1 promotes the cholesterol and phospholipid efflux from peripherial cells.The promoter of ATP-binding cassette transporter A1 gene was cloned into pGL3B-neo vector containing luciferase gene and neomycin resistance gene.The recombinant reporter gene vector pGL3B-neo/ABCA1 was then transfected into hepG2 cells and therefore the stable cell line SABCA1 was obtained.Using this cell-based drug screening system,more than 100 herbal extracts were screened.Through luciferase activity testing,four extracts,shown more than 180% relative activity on SABCA1 cells,were regarded as active samples and the water extract of Euonymus alatus(Thunb.) Sieb.was identified and its EC_(50) value was tested.Furthermore,an apoA1 gene based drug screening system was also used to confirm that the water extract of Euonymus alatus(Thunb.) Sieb.is capable of activating apoA1 gene expression.This herbal medicine extract is eikely to be an effective novel medicine for atheromatosis.