以PRDM1基因启动子为靶点的药物筛选模型的建立研究

摘要

Objective To construct a double luciferase reporter gene vector with PRDM1 gene promoter as target,and establish drug screening cell model in vitro,hope to find small molecule compounds in Chinese herbal medicine library by this model.Methods Total DNA was extracted from 293T cells and it was used for amplifying the fragment contained PRDM1 gene promoter (267 bp/1 257 bp) by PCR.The amplified product was inserted into pGL3-Basic vector.The PCR product and pGL3-PRDM1 vector were verified by sequencing and alignment.The pGL3-PRDM1 and pRL-TK vector were co-transfected into engineer cells.The activity of PRDM1 gene promoter could be assayed by measuring luciferase.The method was optimized by changing ratio of two vectors.Results The highest transfection efficiency and luciferase activity were found in ratio of n(pGL-PRDM1) ∶ n(pRL-TK)=2 ∶1,and with the recombinant luciferase report gene vector contained the length of 1 257 bp amplified fragment transfecting into U266 cells.Moreover,the inductor (5-Aza-CdR) of PRDM1 gene was used for authenticating the method(P＜0.01),the fluoresscence in tensity and promoter activity of PRDM1 gene were enhanced in a dose dependent manner with 5-Aza-CdR.The activity of the promoter of PRDM1 gene was significantly decreased from the concentration of 5 μmol/L of Artemisinid(P＜0.05).The total glucosides of paeoniflorin promoted the promoter activity of PRDM1 gene at a low concentration,and inhibited the promoter activity of PRDM1 gene at a high concentration.Artesunate has no effect on cell proliferation.The effect of total glucosides of paeony on cell proliferation was more complicated.Conclusion A drug selection model targeting PRDM1 gene promoter has been successfully established,and artesunate has been screened to inhibit the promoter activity of PRDM1 gene.%目的 以PRDM1基因启动子为靶点构建双荧光素酶报告基因载体,建立体外药物筛选细胞模型,并对中草药小分子化舍物进行筛选.方法 将人PRDM1基因启动子序列(267、1 257 bp)克隆入荧光素酶报告基因载体pGL3-Basic中,构建重组质粒pGL3-PRDM1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PRDM1基因启动子的启动转录活性,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 成功构建了荧光素酶报告载体pGL3-PRDM1.用0.5、1、2、4μmol/L的5-Aza-CdR处理重组的U266细胞筛选模型,与0μmol/L 5-Aza-CdR相比,增加5-Aza-CdR的刺激,荧光强度及PRDM1基因启动子的活性呈剂量依赖性增强.与0μmol/L相比,青蒿琥酯从5 μmol/L浓度开始对PRDM1基因启动子活性呈明显降低(P＜0.05),在20 μmol/L浓度时降到最低;白芍总苷呈现小剂量促进PRDM1启动子活性,高剂量抑制PRDM1基因启动子活性.青蒿琥酯对细胞生长增殖影响较小,白芍总苷对细胞生长增殖的影响较复杂.结论 成功建立了以PRDM1基因启动子为靶点的药物筛选模型,并筛选出青蒿琥酯能抑制PRDM1基因启动子活性.