剪切修复基因D通过下调mTOR/LOX-1抑制ox-LDL诱导的人脐静脉平滑肌细胞增殖

Xeroderma pigmentosum group D gene inhibits the proliferation of human umbilical vein smooth muscle cell induced by ox-LDLvia down-regulating mTOR/LOX-1 pathway

[目的]探讨剪切修复基因D(XPD)在氧化型低密度脂蛋白(ox-LDL)促血管平滑肌细胞增殖中的作用及分子机制。[方法]将重组质粒pEGFP-N2/XPD利用脂质体转染人脐静脉平滑肌细胞(HUVSMC),沉默哺乳动物雷帕霉素靶蛋白(mTOR)基因,用MTT、EdU法测定各组细胞的增殖;流式细胞仪检测各组凋亡率;利用Western blot检测XPD、血凝素样氧化型低密度脂蛋白受体1(LOX-1)、mTOR、p-mTOR、Bcl-2及Bax的表达。[结果]与对照组比较,ox-LDL组XPD表达明显下调(P<0.05),Bax蛋白表达下调(P<0.05),Bcl-2蛋白、Bcl-2/Bax、mTOR磷酸化活性及LOX-1蛋白表达升高(P<0.05);MTT、BdU结果显示,ox-LDL组细胞增殖较对照组上调(P<0.05)。转染pEGFP-N2/XPD重组质粒能上调Bax蛋白表达(P<0.05)并抑制Bcl-2蛋白、mTOR磷酸化活性及LOX-1蛋白表达(P<0.05),Bcl-2/Bax比值下调(P<0.05);流式细胞仪检测结果显示,转染pEGFP-N2/XPD重组质粒后,细胞凋亡率较对照组增加(P<0.05);MTT、BdU结果显示,转染pEGFP-N2/XPD重组质粒后,细胞增殖较对照组下调(P<0.05)。沉默mTOR基因后,与对照组相比,siRNA mTOR组HUVSMC中LOX-1蛋白表达被抑制(P<0.05)。[结论]XPD能抑制HUVSMC的增殖并且促进其凋亡,下调mTOR和LOX-1蛋白表达,抑制ox-LDL的促HUVSMC增殖和抗凋亡作用,有可能成为抗动脉粥样硬化治疗的靶点。

Aim To investigate the mechanism and signal pathways of xeroderma pigmentosum group D(XPD)gene on the proliferation of human umbilical vein smooth muscle cell(HUVSMC)induced by ox-LDL.Methods HUVSMCs were transfected with the plasmids of pEGFP-N2/XPD using Lipofectamine 2000,and subsequently silent mTOR gene.MTT and EdU assay was used to detect the cell proliferation.Flow cytometry was used to examine the cell apoptosis.The expression of XPD,lectin-like oxidized low-density lipoprotein receptor 1(LOX-1),mTOR,phosphomTOR,Bcl-2 and Bax was measured by Western blot.Results The expression of XPD and Bax protein was downregulated in ox-LDL group(P<0.05),while the expression of LOX-1,mTOR,Bcl-2 protein and the ratio of Bcl-2/Bax was significantly up-regulated(P<0.05),compared with control group.Cell proliferation of ox-LDL group increased obviously(P<0.05).After transfected with the pEGFP-N2/XPD plasmid,the expression of Bax was significantly up-regulated,while the expression of LOX-1,mTOR,Bcl-2 and the ratio of Bcl-2/Bax were significantly down-regulated(P<0.05).Flow cytometry showed that overexpression of XPD increased the apoptosis rate of HUVSMC(P<0.05).MTT and BdU showed that cell proliferation of pEGFP-N2/XPD group reduced compared with control group(P<0.05).Com-pared with control group,the expression of LOX-1 was significantly down-regulated in siRNA mTOR group(P<0.05).Conclusion XPD can inhibit HUVSMC proliferation and promote its apoptosis,and reduce the effect of ox-LDL promoting proliferation of HUVSMC via the mTOR/LOX-1 pathway.XPD may be the target of treatment of atherosclerosis.