人剪切修复基因着色性干皮病基因D对人脐静脉内皮细胞的促凋亡作用

目的探讨人剪切修复基因着色性干皮病基因D(XPD)对人脐静脉内皮细胞(HUVEC)的促凋亡作用。方法用脂质体转染法瞬时转染HUVEC,转染重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2,并用未转染的与重组质粒pEGFP-N2/XPD和空载质粒pEGFP-N2具有相同遗传背景和代数的HUVEC作为空白对照。实验分为3组:正常对照组、pEGFP-N2组和pEGFP-N2/XPD组。用荧光显微镜观察绿色荧光蛋白报告基因表达情况,用流式细胞仪检测细胞凋亡情况,用RT-PCR和Western Blot检测XPD、Bcl-2、Bax和wt-p53表达量的变化,用MTT法观察细胞增殖活力。结果在荧光显微镜下,可在转染了重组质粒pEGFP-N2/XPD或空载质粒pEGFP-N2的细胞中观察到绿色荧光,即转染成功;流式细胞仪结果显示,重组质粒pEGFP-N2/XPD的转染引起细胞凋亡增加(P<0.05或P<0.01);RT-PCR和Western Blot检测发现,重组质粒pEGFP-N2/XPD的转染使得XPD表达增高(P<0.05),同时使得Bcl-2表达降低,Bax和wt-p53表达增高(P<0.05或P<0.01);MTT结果显示,重组质粒pEGFP-N2/XPD的转染抑制细胞增殖活力(P<0.05)。结论 XPD能促进HUVEC凋亡,下调XPD的表达,有望成为治疗动脉粥样硬化的一个新靶点。

The Promoting Effects of Xeroderma Pigmentosum D Gene on Proliferation of Human Umbilical Vein Endothelia Cells

AimTo investigate pro-apoptosis effects of xeroderma pigmentosum D(XPD) gene on human umbilical vein endothelia cells(HUVEC).MethodsRecombinant plasmid pEGFP-N2/XPD and vacant vector plasmid pEGFP-N2 were transient transfected into HUVEC by liposome 2000 method,with the same genetic background and algebra HUVEC as blank controls.The experiments were divided into three groups:control group,pEGFP-N2 group and pEGFP-N2/XPD group.The expression of green fluorescent protein was observed through fluorescence microscopy;the cell apoptosis rate was examined by flow cytometry;through RT-PCR and Western Blot,the expression levels of XPD,Bcl-2 and Bax and wt-p53 were detected;the cell growth was detected by MTT.ResultsGreen fluorescences were observed in the cells transfected with pEGFP-N2/XPD or pEGFP-N2,indicating that the plasmids were transfected successfully.Flow cytometry results showed that overexpression of XPD increased the apoptosic rate of HUVEC(P<0.05 or P<0.01).RT-PCR results and Western Blot results showed that the transfection of pEGFP-N2/XPD increased the expression of XPD,Bax and wt-p53(P<0.05),decreased the expression of Bcl-2(P<0.05);MTT results showed that the transfection of pEGFP-N2/XPD inhibited the cell growth(P<0.05).ConclusionsXPD gene can promote HUVEC apoptosis.Therefore,down-regulating the expression of XPD gene is likely to be potential molecular target for treatment of atherosclerosis.