大鼠骨髓间充质干细胞体外诱导分化为具有起搏功能的心肌细胞

Differentiation of rats bone marrow mesynchymal stem cells into cardiomyogenic cells with pacemaking function

目的寻求体外诱导骨髓间充质干细胞分化为具有起搏功能的心肌细胞的方法和途径。方法分离大鼠骨髓间充质干细胞,在体外分别用5-氮胞苷(5AZA)、碱性成纤维细胞生长因子(bFGF)+内皮细胞生长因子(EGF)、肝细胞生长因子(HGF)、干细胞因子(SCF)和窦房结心肌细胞(SAN CMs)裂解液进行诱导。观察细胞形态,用免疫组化和流式细胞技术测定心脏特异性肌钙蛋白T(cTnT)、连接蛋白43以及超极化激活的核酸环化酶门控的离子通道(HCN)2/4的表达,并测定细胞起搏电流If。结果上述方法均能在体外将干细胞诱导为表达心肌细胞特异性蛋白的心肌样细胞,并且均有HCN2表达。5-AZA、bFGF+EGF和SAN CMs组表达HCN2比例较高(22.9%、22.3%和11%),且均能测到起搏电流(If)的存在。结论窦房结裂解液是体外诱导骨髓间质干细胞分化为具有起搏功能的心肌细胞的理想方法,HCN是很有前景的分选具有起搏功能的心肌细胞的标志蛋白。

Objective To find the way of inducing cells with pacemaking function in vitro. Methods the bone marrow mesynchymal stem cells（MSCs） into cardiac Dissociate the rat MSCs and induce them with 5AZA, bFGF ＋ EGF, HGF, SCF and lysate of the sinoatrial cells respectively. The morphological changes were observed,and the expressing of protein cTnT, connexin 43 and HCN2/4 were analyse by immunohistologic and fiowcytometry techniques. The pacmaking current If were evaluted by patch clamp techniques. Results All the methods can induce the bone marrow MSCs to differentiate into cardiac cells, which expressing cardiac cell specific protein and HCN2. Cells induced by 5AZA, bFGF ＋ EGF and SAN CMs show higher rate of HCN2 expressing （22.9% , 22.3% , 11% ）. The cells of these groups have the pacmaking current If. Conclusion Lysate of the sinoatrial cells are ideal methods of inducing the bone marrow MSCs to differentiate into cardiac cells with pacemaking function in vitro. HCN is a promising marker protein to select pacmaking cells out of the differentiated cells.