骨髓间充质干细胞体外分化为心肌样细胞相关调控基因的时序表达

Temporal expression of regulative genes in the process of bone marrow mesenchymal stem cells differentiating into cardiomyocytes in vitro

目的研究体外诱导骨髓间充质干细胞(MSCs)向心肌样细胞分化过程中相关调控基因的时序表达,筛选MSCs定向分化为心肌细胞的重要调控基因。方法采用第8代的MSCs,以去甲基化药物5氮杂胞苷进行体外诱导,连续观察8周,相差显微镜下观察其形态变化,荧光免疫组化方法鉴定心肌特征性蛋白肌球蛋白重链(MHC)和连接蛋白Connexin43的表达,应用半定量RTPCR技术分析TGFβ、Nkx25、GATA4、MEF2C、TEF1和RARα等相关调控基因在分化过程中的动态时序表达。结果诱导前MSCs呈成纤维细胞样,诱导后细胞形态发生变化,1周呈棒状或球形,2周后细胞之间形成连接,排列方向渐趋一致,3周时开始出现肌管结构。MHC诱导前无表达,诱导后1周开始表达,2周起表达逐渐增强,8周时阳性细胞比例为30%;Connexin43在诱导前有弱表达,阳性率为5%,诱导后逐渐增强,8周时阳性细胞比例为40%。TGFβ、Nkx25、GATA4和MEF2C基因在诱导后1天表达开始增强,诱导后1周达高峰,以后维持在高水平;TEF1和RARα基因在诱导过程中表达无明显变化。结论TGFβ、Nkx25、GATA4和MEF2C可能是调控MSCs定向分化为心肌样细胞的重要调控基因。

Objective To investigate the temporal expression of regulative genes in the process of bone marrow mesenchymal stem cells (MSCs) differentiating into cardiomyocytes in vitro and screen some important genes in this differentiation. Methods MSCs were isolated and expanded as undifferentiated cells in culture for more than 8 passages. MSCs were treated with 10 μmol/L 5 azacytidine for 24 hours and were maintained for eight weeks. The differentiation of MSCs was observed by phase contrast microscope, fluorescence immunohistochemistry and semi quantitative RT PCR from one week to eight weeks. Results MSCs showed a fibroblast like morphology, but the morphology changed after induction. They formed a ball like or stick like morphology at 1 week, connected with adjoining cells, aligned in a striated pattern after 2 weeks and formed myotube like structure at 3 weeks. Myosin heavy chain (MHC) began to be expressed at 1 week after induction and significant from 2 weeks after induction. The rate of positive cells was 30% at 8 weeks after induction. Connexin43 was expressed weakly before induction and significantly after that. The rate of positive cells was 40% at 8 weeks after induction. The expression levels of TGF β, Nkx 2.5, GATA 4 and MEF 2C genes were low before induction, began to increase at 1 day after induction, reached the peak at 1 week and kept high expression level after 1 week. The expression level of TEF 1 and RARα kept unchanging. Conclusion TGF β, Nkx 2 5, GATA 4 and MEF 2C genes probably play an important role in the process of MSCs differentiating into cardiomyocytes.