重组腺病毒Vpr诱导脑胶质瘤细胞凋亡的体外研究

An experiment study about the apoptosis effect induced by adenovirus mediated HIV1-Vpr on human gUoma cells in vitro

目的 探讨重组腺病毒介导的人类免疫缺陷病毒1型病毒蛋白R（HIV1-Vpr）体外对胶质瘤细胞U251凋亡的影响.方法 将U251细胞分为正常对照组、空载体组和实验组进行细胞培养,24 h后空载体组和实验组按MOI=50分别进行空载体腺病毒（rAd5-null）和含有HIV1-Vpr基因的重组腺病毒（rAd5-Vpr）转染,转染后用MTT比色法测定细胞增殖活性,用Hoechst染色和流式细胞仪分别检测细胞的凋亡和细胞周期改变,用Western blot方法 检测相关蛋白的表达.结果 rAd5-Vpr能够抑制U251细胞增殖,其作用从转染后48 h开始,随着时间的延长,抑制作用逐渐增强（P＜0.05）;实验组Hoechst染色观察到明显的细胞凋亡现象;流式细胞周期检测显示实验组细胞G2期比例升高（P＜0.05）;Western blot结果 显示,实验组U251细胞经转染rAd5-Vpr72 h后,可见Vpr蛋白表达,同时Bcl-2蛋白表达降低,Bax、Caspase3和Fas-L蛋白表达增强.结论 rAd5-Vpr在体外能够抑制U251细胞增殖,诱导细胞周期G2期阻滞和细胞凋亡.

Objective To study the effect of recombinant adenovirus mediated transfection of human immunodeficiency virus type 1 viral protein R （HIV1 - Vpr） on apoptosis of glioma U251 cells in vitro.Methods U251 cells under culture were divided into the normal control group, mock group and experiment group.After 24 hours of culture, the mock and experiment group were transfected with mock vector（rAd5 -null）and the recombinant adenovirus carrying HIV1 -Vpr （rAd5 - Vpr） respectively at a multiplicity of infection （MOI） of 50.The proliferation activity of cells was detected by MTT assay, the apoptosis effect and the change of cell cycle were determined by Hoechst stain and flow cytometry （FCM） technology respectively. The expression of related proteins was revealed by the method of Western blot. Results rAd5 - Vpr could inhibit the proliferation of U251 cells, and this effect may start48 hours after transfection and increase with the prolonged time （P 〈 0.05）. The apoptosis effect was observed obviously by Hoechst stain, and by FCM there showed the ratio of G2 cycle cells was significantly high in the experiment group（P 〉 0.05）; The results of Western blot showed that after 72 hours of transfection the Vpr protein expression was observed, meanwhile, the Bcl-2 protein showed decreased expression while in the Bax,Caspase3 and Fas - L proteins increased expression Conclusion rAd5 - Vpr could inhibit the proliferation of U251cells and induce cell cycle G2 arrest and apoptosis in vitro.