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

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

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。方法培养小鼠骨髓间充质干细胞并利用血小板源生长因子（PDGF—BB，50mg／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，48h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（SMmyosinheavychain，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myocardinmRNA表达下调42．86％，差异有统计学意义（P〈0．01）；且免疫组织化学结果表明：干扰组SM—MHC阳性表达低于空白对照组（P〈0．01）。结论小鼠骨髓间充质干细胞中存在少数细胞具有分化成为平滑肌样细胞的潜能；构建的pGen-myo—shRNA重组质粒能有效抑制myocardin基因的表达，且对小鼠骨髓间充质干细胞向平滑肌样细胞分化有抑制作用。

Objective Construction of a small interfering RNA （siRNA） eukaryotic expression vector specific to mouse myocardin gene and study on the role of myocardin-siRNA on differentiation of mouse bone mesenchymal 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 transfected 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 successfully and the myocardin mRNA was reduced 42. 86% by pGen-myoshRNA in comparing with that of the controls （ P 〈 0. 01 ） ; and the expression of SM-MHC protein was downregulated （P 〈 0. 01 ）. Conclusion Subset of mouse MSCs have the potential to differentiate into smooth muscle-like cells, a possible cell source responsible for atherosclerotic plaque formation, and myocardin expression may play an important role during this process.