大鼠骨髓间充质干细胞体内分化为心肌细胞的相关基因

Relative gene in rat mesenchymal stem cells differentiated into cardiomyocyte in vivo

目的研究大鼠骨髓间充质干细胞(MSCs)在体内诱导分化为心肌细胞的过程中相关调控基因的时序表达,揭示MSCs体内分化为心肌细胞的分子调控机制。方法从大鼠骨髓中分离MSCs并传至第8代,用DAPI标记后注射到正常大鼠心肌组织内,分别于细胞移植后1 d、1、2、3、4、6和8周处死大鼠,在注射点获取心肌标本;采用HE染色观察MSCs植入后的形态变化,荧光免疫组化检测心肌特异性蛋白MHC和connexin43的表达;半定量RT-PCR检测TGF-β、Nkx-2.5、GATA-4、MEF-2C、TEF-1和RARα等相关调控基因的动态时序表达。结果MSCs注入正常心肌组织后可逐渐向心肌细胞转化,从小圆形未分化细胞逐渐转变为与周围宿主组织连接的心肌样细胞;移植1周后出现MHC的表达,移植4周后宿主心肌细胞与移植细胞间出现connexin43的表达,Nkx-2.5和GATA-4基因诱导后1 d表达开始增强,1周时达高峰,以后维持在高水平,TGF-β、MEF-2C、TEF-1和RARα基因mRNA在诱导前后无明显变化。结论Nkx-2.5和GATA-4基因在MSCs体内转化为心肌细胞的过程中可能发挥重要作用。

Objective To investigate the temporal expression of genes associated with bone marrow mesenchymal stem cells （MSCs） differentiation into cardiomyocytes. Methods MSCs were injected into the myocardium of normal hearts. The hearts were harvested after implantation. The change of MSCs morphology was observed by HE staining. The expression of cardiac-specific proteins myosin heavy chain（MHC） and connexin43 were detected by fluorescence immunohistochemistry. Temporal expression of some regulative genes mRNA was detected by semi-quantitative RT-PCR. Results MSCs gradually differentiated into cardiomyocytes from small, round, and undifferentiated cells in close approximation to intact host cardiomyocytes after grafted into normal myocardium. MHC-positive cells were detected at 1 week and connexin43 were found in the space between the host cardiomyocytes and transplanted MSCs after 4 weeks. The expression level of Nkx-2.5 and GATA-4 genes began to increase at day 1 of treatment, reach the peak at 1 week and kept high expression level after 1 week. The expression level of TGF-β、 MEF-2 C、 TEF-1 and RARa mRNA kept invariant. Conclusion Nkx-2.5 and GATA-4 genes probably play an important role in the process of MSCs differentiation into cardiomyocytes.