myocardin基因沉默在PDGF-BB诱导小鼠骨髓间充质干细胞向平滑肌样细胞分化中的作用

目的 构建myocardin特异性siRNA真核表达载体,体外观察其对myocardin基因的沉默效应及对小鼠骨髓间充质干细胞(MSC)向平滑肌样细胞分化过程中的影响.方法 培养小鼠骨髓问充质干细胞并利用血小板源生长因子(PDGF-BB,50 mg/L)联合高浓度胎牛血清(20%FBS)诱导其向平滑肌样细胞分化;构建含U6启动子和myocardin特异短发卡RNA(shRNA)编码序列的质粒载体pGen-myo-shRNA,以含非特异性shRNA编码序列的质粒载体pGenesil-Con(pCon)为阴性对照,分别转染诱导培养后第6天的小鼠MSC,48 h后用RT-PCR技术检测细胞内myocardin在mRNA水平的表达;同时用免疫组织化学方法 检测平滑肌肌球蛋白重链(sM myosin heavy chain,SM-MHC)的表达来鉴定平滑肌样细胞的分化.结果 成功构建myocardin特异性siRNA真核表达载体,利用其沉默myocardin基因后,干扰组相比空白对照组myocardin mRNA表达下调42.86%,差异有统计学意义(P

Effect of silencing myocardin gene expression on differentiation of mouse bone mesenchymal stem cells into smooth muscle-like cells induced by PDGF-BB

Objective Construction of a sinall interfering RNA(siRNA)eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesonchymal stem cells(MSCs) into smooth muscle-like cells induced by PDGF-BB in vitro. Methods Mouse MSCs were isolated from bone marrow and cultured with 50 mg/L PDGF-BB and fetal bovine serum(20%).Specific myocardin-siRNA sequence was cloned into a plasmid pGenesil-1.0 vector. which contained U6 promoter.The recombinant plasmid and control plasmid were transfeeted into MSCs which had been cultured with PDGF-BB for 6 days beforehand.The expression of myocardin mRNA was detected by RT-PCR 48 hours after the transfection.Immunohistochemistry Was used to detect the SM-MHC and tO identify the smooth muscle-like cells.Results The recombinant plasmids carrying myocardin-siRNA sequences were constructed sueeessfully and the myocardin mRNA was reduced 42.86% by pGen-myo- shRNA in comparing with that of the controls(P<0.01);and the expression of SM-MHC protein was down- regulated(P<0.01).Conclusion Subset of mouse MSCs have the potential to differentiate into smooth muscle-like cells,a possible cell source responsible for atheroselerotic plaque formation.and myocardin expression may play an important role during this process.