基于报告基因和PPARγ信号通路的药物筛选模型的建立

Development of drug screening model based on reporter gene and the signal transduction of PPARγ

目的 建立基于报告基因和PPARγ(peroxisomeprolif erator activatedreceptorγ)信号通路的药物筛选模型,用此模型筛选具有胰岛素增敏活性的小分子化合物。方法 五种细胞分别进行瞬时转染,将含有目的片段PPRE(peroxisomeproliferatorresponseelement)和报告基因荧光素酶(Luc)的质粒及表达PPARγ的质粒共转染到细胞中,通过测定荧光素酶活力来考察马来酸罗格列酮对PPARγ信号通路的影响,选取诱导表达倍数最高的细胞株建立模型。用其他类型核受体激动剂对此模型进行特异性考察。结果 293T细胞中,荧光素酶的表达受马来酸罗格列酮的诱导倍数最高,可达4 9倍,并呈现一定的剂量依赖关系,Z′因子为0 .72。而其他各类核受体激动剂的诱导表达率均在1倍左右。马来酸罗格列酮在转染剂量范围内无促进细胞增殖的作用。结论 马来酸罗格列酮对共转染报告基因质粒和表达PPARγ质粒的293T细胞Luc的表达具有较强的诱导作用,此模型具有较好的特异性和稳定性,适用于建立筛选PPARγ激动剂的高通量筛选模型。

Aim To establish a method of drug screening based on reporter gene and the signal transduction of PPARγ(Peroxisome proliferator-activated receptor γ) system for discovering the small molecular compounds with activity of improving insulin sensitivity.Methods A recombinant vector PPRE-tk-Luc^+ was transiently cotransfected with a PPARγ expression vector pCMX- PPARγ into five cell lines respectively. The effect of rosiglitazone on the signal transduction of PPARγ was determined by examing the luciferase activity, the cell line with the highest induction rate was selected for further experiments. The speciality of induction was examined by the ligands of nuclear receptors.Results The expression levels of reporter gene induced by rosiglitazone in 293T cells was the highest, up to 4.9 fold, and in dose dependent manner. The Z'factor was 0.72. The expression levels of luciferase in 293T cells induced by the ligands of nuclear receptors were about 1.0 fold. No effect of rosiglitazone on the proliferation of 293T was observed.Conclusions The expression of luciferase in 293T cells co-transfected with the recombinant vector PPRE-tk-Luc^+ and pCMX-PPARγ vector could be induced significantly. The preferable speciality and stability of the cellular model make it well suited for establishing high-throughput screening systems for agonists of PPARγ.