微小RNA可在人骨髓间充质干细胞分化来源的心肌样细胞中表达

MicroRNAs can be expressed in cardiomyocyte-like cells differentiated from human mesenchymal stem cells

目的研究人骨髓间充质干细胞(hMSCs)分化来源的心肌样细胞中代表性微小RNA(miRNAs)的表达。方法利用FITC-偶联的CD29、CD34和CD11b抗体在流式细胞仪上检测分离的hMSCs的抗原表型。分别用5-氮胞苷(5-aza)和乳鼠心肌非接触共培养法诱导传至3代的hMSCs分化为心肌样细胞。用免疫化学法检测hMSCs分化来源的心肌样细胞中心肌特异的α-肌节辅肌动蛋白和心肌肌钙蛋白(cTnI)的表达。利用逆转录PCR和DNA测序鉴定5个代表性的心脏特异的初级微小RNA(pri-miRNAs)的表达。结果hMSC标志分子CD29表达率为98.87%,而造血细胞标志分子CD34和CD11b的表达率只是5%和0.4%。免疫化学分析显示,分别经5-aza和乳鼠心肌非接触共培养诱导的hMSC均可表达α-肌节辅肌动蛋白和cTnI,而在未分化的hMSCs中无表达。miRNA-143,-181可在5-aza诱导的hMSCs中表达,miRNA-143,-181,-206,-208可在乳鼠心肌非接触共培养的hMSCs中表达,而两种诱导方法均未能诱导miRNA-1-2在hMSCs中表达。结论利用5-aza和乳鼠心肌非接触共培养法诱导hMSCs分化的心肌样细胞中可以表达不同的心脏特异的pri-miRNAs。

Objective To investigate the expression of representative heart-specific primary microRNAs （pri-miRNAs） in the cardiomyocyte-like cells differentiated from human mesenchymal stem cells （hMSCs）. Methods The phenotype ofhMSCs isolated was identified by flow cytometry using monoclonal antibodies against FITC-conjugated CD29, CD34, and CD1 lb. The third-passage hMSCs were induced to differentiate into cardiomyocyte-like cells by 5-azacytidine and indirect coculture with neonatal rat myocytes, respectively. Immunocytochemical analysis was performed to detect the expression of the cardiac- specific proteins, namely cardiac troponin I （cTnI） and sarcomeric ct-actinin, in the cardiomyocyte-like cells differentiated from hMSCs. RT-PCR and DNA sequencing were used to identify the expression of the 5 representative heart-specific pri-miRNAs. Results High hMSC marker CD29 expression rate （98.87%） and low hematopoietic cell markers CD34 （5%） and CD1 lb （0.4%） expression rates were identified in the hMSCs isolated, cTnI and sarcomeric ct-actinin expression occurred in the hMSCs following induction with the 2 differentiation-inducing methods, miRNA-143 and -181 expressions were induced in the hMSCs by 5-azacytidine and miRNA-143, -181, -206, and -208 expressions were induced by indirect coculture with neonatal rat myocytes, but pri-miRNA-1-2 expression failed to be induced by these two induction methods. Conclusion Expressions of the representative heart-specific pri-miRNAs in different patterns can be induced in cardiomyocyte-like cells differentiated from hMSCs by 5-azacytidine and indirect coculture with neonatal rat myocytes.