胚胎干细胞在心肌样环境中向心肌样细胞的分化情况

目的探讨心肌细胞促进胚胎干细胞(embryonic stem cells,ESCs)分化为心肌样细胞的诱导作用。方法收集小鼠3.5d胚龄的囊胚,将其培养在小鼠胚胎成纤维细胞饲养层上,4～5d后取内细胞团接种在饲养层上分离培养出ESCs。取3～5代ESCs,先将ESCs悬浮培养形成2～3d的拟胚体(embryoid bodies,EBs),再与新生大鼠心肌细胞共培养诱导向心肌细胞分化,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T(TnT)、а-肌动蛋白(а-Actin)的表达。结果诱导d3起可见自发性、有节律跳动的拟胚体出现,12d时共培养组约有93%的拟胚体出现节律性收缩,显著高于对照组,均表达心肌细胞特异性蛋白cTnT、а-actin,心肌细胞直接接触诱导组其分化比率达56.5%,分化的细胞形态较单一。结论心肌细胞与胚胎干细胞直接接触能促进胚胎干细胞向心肌细胞分化。

Differentiation of mouse embryonic stem cells into cardiomyogenic cells in cardiomyocytes

OBJECTIVE To explore the effect of embryonic stem cells (ESCs) differentiating into cardiomyocytes (CM) in cardiomyocytes. METHODS The blastocytes of 3.5d were collected from Kunming species mouse, cultured on mouse embryonic fibroblaste cell feeder layers for 4-5d, and the ESCs were isolated and subsequently cultured in vitro. At the day of 2 to 3, the embryoid bodies (EBs) were derived from ESCs of passage 3 to 5, and then cocultured with neonatal cardiomyocyte (CM) to induce into cardiomyocytes. Then the expression of cardiac specific cardiac troponin-T (TnT) and а-Actin were detected by immunofluoresence. RESULTS The rhythmic beating embryoid bodies were observed on day 3, and reached to 93% of rhythmic beating embryoid bodies on the 12th day in cocultured group. All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for TnT and а-Actin, and the group induced by direct cell to cell contact acquired the highest differentiating ratio of 56.5%, and was the highest differentiating ratio compared with other groups (P﹤0.05). CONCLUSION Car- diomyocyte coculturing system and direct cell-to-cell interaction can induce mouse ESCs to differentiate into cardiomyocyte-like cells.