大鼠骨髓间充质干细胞体外诱导分化为心脏起搏样细胞的研究

Study of differentiation of rat mesenchymal stem cells with mHCN4 gene into spontaneous cardiomyocyte-like cells in vitro

目的探讨超极化激活的环核苷酸门控通道亚型4(mHCN4)基因修饰对大鼠骨髓间充质干细胞(rM-SCs)体外定向分化为心脏起搏样细胞的影响。方法以构建的mHCN4逆转录病毒载体转染第三代rMSCs,获取稳定高效表达mHCN4基因的细胞,观察细胞形态学改变,并进行α-actin、TroponinT、HCN4、Connexin43等基因表达检测。结果rMSCs转染mHCN4基因后,可获得高效稳定表达mHCN4的细胞,经连续不传代培养后出现大量的肌管样分化,并出现自主搏动的细胞,α-actin、TroponinT、HCN4表达检测均为阳性;而只转染绿荧光蛋白(GFP)的rMSCs仅出现少量的肌管样分化,并未观察到细胞的自主搏动;未转染组MSCs则未观察到肌管样分化。结论mHCN4基因修饰的rMSCs可在体外定向分化为具有自律性的心脏起搏样细胞,而mHCN4基因活化可能是促进rMSCs向心脏起搏样细胞分化的关键因素。

Objective To study the role of mHCN4 gene expression in differentiation of rat mesenchymal stem cells （rMSCs） into spontaneous cardiomyocyte-like cells in vitro. Methods rMSCs from Sprague-Dawley （SD）rats were cultured and passed repeatedly to P3. Experimental groups were divided by transfected with retrovirus vectors expressing mHCN4- EGFP or only EGFP. The continuous morphological changes of the cells were observed with inverted microscope after the treatment. The expression of α-actin, Troponin T, HCN4 was determined by immunostaining. Results rMSCs expressed mHCN4 gene steadily after transfection with mHCN4- EGFP. After cultured 20 days without subculture, rMSCs expressing mHCN4 formed abundance myotube-like structures , showed spontaneously beating and positive expression of α-actin, Troponin T and HCN4. But in rMSCs only expressing GFP, a few myotube-like structures and no spontaneously beating were observed. Conclusion rMSCs with mHCN4 gene can differentiate into spontaneous cardic pacing cells in vitro. Activation of mHCN4 gene probably play an important role in differentiate into cardic pacing cells of MSCs.