microRNA499慢病毒载体转染诱导大鼠骨髓间充质干细胞向心肌样细胞分化

miR-499 Induces Cardiac Differentiation of Rat Bone Marrow-Derived Mesenchymal Stem Cells

目的:探讨microRNA 499(miR-499)慢病毒转染对诱导大鼠骨髓来源间充质干细胞(BM-MSCs)向心肌样细胞分化的作用。方法:取第四代Wistar大鼠骨髓来源间充质干细胞进行流式细胞检测,鉴定干细胞表面特异标记物。使用符合干细胞鉴定标准的细胞批次用于后续实验。实验设置miR499慢病毒转染、慢病毒空白转染2个处理组,分别于处理后即日、1d,3d,5d,7d收集细胞进行下列实验:实时荧光定量PCR检测心肌重要转录因子GATA4、NKx2.5和MEF2C的mRNA表达,western-blot检测心肌特异蛋白I(cTnI)的表达。结果:培养第四代Wistar大鼠骨髓来源间充质干细胞表达干细胞表面特异标记物,可用于实验。大鼠骨髓来源间充质干细胞microRNA 499慢病毒载体转染后microRNA 499表达明显升高,且转染后1d,3d,5d,7d,GATA4、NKx2.5和MEF2C的mRNA表达逐渐增强。慢病毒空白转染组未见明显变化。western-blot检测自第3天开始可见cTnI阳性表达条带,慢病毒空白转染组未检测到明显阳性表达条带。结论:microRNA 499可诱导大鼠骨髓来源间充质干细胞向心肌样细胞分化。

Objective： To investigate the effect of miR-499 on differentiation of cardiomyocyte-like cells from rat bone marrow-derived mesenchymal stem cells （BM-MSCs）. Methods： The fourth passage of rat BM-MSCs were infected with lentiviral vectors bearing miR-499 or empty lentiviral vectors as control. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by real-time PCR and western blot. Results： The fourth passage of the cultured rat BM-MSCs express significant markers of MSCs. After transfection of microRNA 499 in rat BM-MSCs, the expression level of microRNA 499 was markedly enhanced. The mRNA expression of GATA4, NKx2.5 and MEF2C gradually increased from day 1 to day 7, the cTnI expression was detected from day 3. While there were no significant changes of GATA4, NKx2.5 and MEF2C mRNA levels in BM-MSCs infected with empty lentiviral vectors, and cTnI expression cannot be detected either. Conclusion： Over-expression of miR-499 rat BM-MSCs can induce rat BM-MSCs toward cardiac differentiation.