以Insig-2基因启动子为靶点的药物筛选模型的建立

Establishment of drug screening model targeting Insig-2 promoter

目的通过双荧光素酶报告基因检测系统建立以胰岛素诱导基因2(insulin-induced gene 2,Insig-2)启动子为靶点的药物筛选模型。方法将人Insig-2基因启动子序列克隆入荧光素酶报告基因载体pGL3-basic中,构建重组质粒pGL3-Insig-2并与内参质粒pRL-TK瞬时共转染工具细胞,通过检测荧光素酶报告基因表达水平的变化反映Insig-2基因启动子启动转录的活性,并对共转染质粒比例、工具细胞选择等条件进行探索和优化,相关药物处理进行验证。结果成功构建了重组质粒pGL3-Insig-2;确认pGL3-Insig-2:pRL-TK共转染比例为4∶1,确定3T3-L1细胞为工具细胞;1,25-(OH)2D3、小檗碱和姜黄素均能明显增强Insig-2基因启动子活性。结论成功建立了以Insig-2基因启动子为靶点的药物筛选模型,为筛选新型调脂药奠定基础。

Aim To establish a drug screening model targeting Insig-2 prompter by the dual- luciferase reporter assay system. Methods The Insig-2 promoter fragment was inserted into the vector pGL3-basic. The constructed recombinant plasmid pGL3- Insig-2 and internal reference plasmid pRL-TK were transiently co-transfected to appropriate cells, then the Insig-2 promoter ac- tivity was measured by dual-luciferase reporter assay, and the assay conditions were studied and optimized, such as the ratio of plasmids, the choice of cell lines, and related drug treatment. Results The recombinant plasmid pGL3-Insig-2 was successfully constructed; the ratio between plasmid pGI3-Insig-2 and pRL- TK was 4：1; 333-L1 cell line was chosen as target cell; 1,25- （OH） 2D3, Berberine and Curcumin could significantly enhance the Insig-2 gene promoter activity. Conclusion The drug screening model targeting Insig-2 promoter was successfully es- tablished, which lays an experimental foundation for discovering new lipid-regulators.