心肌细胞介导骨髓间充质干细胞的心肌样分化

Cardiomyocyte-induced myogenic differentiation of marrow mesenchymal stem cells

目的探讨骨髓间充质干细胞(m esenchym al stem cells,MSCs)在不同培养条件下定向分化过程中的生物学特性变化及其机制。方法贴壁法分离大鼠MSCs,培养至第6代。实验分3组:条件培养基组,用新生鼠心肌细胞培养上清液制成的条件培养基处理MSCs 48 h 2次,其间间隔48 h;混合培养组,新生鼠心肌细胞和MSCs按1∶1的比例混合培养1周;对照组,MSCs常规培养。检测各组MSCs中心肌细胞结构功能蛋白:心肌肌联蛋白(titin)、肌球蛋白重链(myosinheavy chain,MHC)、心肌组织缝隙连接的特异性组成蛋白质Connexin43(Cx43)的表达情况。结果①条件培养基可诱导MSCs心肌样分化,表达心肌细胞骨架蛋白titin的Z带及A带部分,Cx43大量表达,但在检测期内未观察到MHC的表达;②MSCs在混合培养中与心肌细胞形成连接,表达titin,部分细胞内可检测到类似肌小节的结构,Cx43集中分布于细胞相邻的界面;③对照组中的部分MSCs可少量表达Cx43及titin。结论源于心肌细胞的各种因素可促使MSCs心肌样分化,分化的程度取决于作用信号的性质。

Objective To investigate the differentiation of marrow mesenchymal stem cells （MSCs） under various in vitro conditions and analyze its mechanisms. Methods Rat MSCs were isolated and passaged. MSCs were either cultured in conditioned medium, the combination of supernatant of cardimyocyte culture and common medium, or cocuhured with neonatal cardiomyocytes at the ratio of 1：1 （cocultured group）, or under normal medium （control group）. After 7 d of induction, immunofluorescence was employed to detect titin, early cytoskeletal protein of cardiomyocyte, myosin heavy chain （ MHC ） and Connexin43 （ Cx43 ） expression in MSCs. Results ①Conditioned medium could induce MSCs into cardiomyocyte lineage. Z part and A part of titin, large quantity of Cx43 could be detected after 1 week while myosin heavy chain was absent; ②MSCs could form junctions with neighboring cardiomyocytes. Above mentioned cardiomyocyte proteins were expressed in cocuhured MSCs. Sarcomeric-like structure was detected in the cytoplasm of cocuhured MSCs. Cx43 was mainly distributed in the MSC-cardiomyocyte interface. ③Untreated rat MSCs expressed myocardial gap junction protein Cx43 and titin in a lower degree. Conclusion Microenvironment of cardiomyocyte can induce the differentiation of MSCs into myocyte phenotype in vitro, either by soluble signaling molecules or by direct cell- to-cell contact. Degree of differentiation depends on the signal properties.