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目的研究淫羊藿苷(icariin,ICA)体外诱导小鼠胚胎干细胞(embryonic stem cells,ESCs)ES-E14细胞分化为心肌细胞的作用。方法复苏的ESCs经直接悬浮法形成拟胚体(EBs),应用ICA定向诱导,相差显微镜下观察分化细胞的形态学变化,免疫细胞荧光技术及Western blot(WB)分别检测心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的蛋白表达及表达量的变化;透射电镜观察分化细胞的超微结构。结果经ICA诱导后第5天的EBs出现了细胞跳动点,ICA诱导后第3天的细胞心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的蛋白表达均阳性,于诱导后的第3天(早期)、第12天(中期)及第20天(晚期)心肌细胞特异性肌钙蛋白I(TnI)、心室肌球蛋白轻链(Mlc-1v)的表达逐渐增多,晚期明显高于早期及中期;透射电镜下可见大量平行排列的肌丝。结论用直接悬浮法,ICA体外能诱导鼠ESCs分化为心肌细胞,分化的心肌细胞两种心肌特异性结构蛋白量的表达随分化进程的进展逐渐增多。
Objective To investigate the effect of icariin (ICA) on differentiation of ES-E14 cells from mouse embryonic stem cells (ESCs) into cardiomyocytes in vitro. Methods ESCs were resuscitated by direct suspension to form embryoid bodies (EBs), and induced by ICA. Morphological changes of differentiated cells were observed under phase contrast microscope. Immunofluorescence and Western blot (WB) were used to detect cardiomyocyte-specific calcium Protein I (TnI) and ventricular myosin light chain (Mlc-1v) protein expression and expression changes; transmission electron microscopy of differentiated cells ultrastructure. RESULTS: The EBs on day 5 after induction by ICA showed cell beating point. The protein expression of cardiomyocyte specific troponin I (TnI) and ventricular myosin light chain (Mlc-1v) on day 3 after induction of ICA Were positive for cardiomyocyte-specific troponin I (TnI), ventricular myosin light chain (Mlc-1v) on day 3 (early), day 12 Gradually increased in the late stage, which was significantly higher than that in the early stage and in the middle stage. A large number of myofilaments arranged parallel to each other were observed under transmission electron microscope. Conclusions ICA can induce the differentiation of murine ESCs into cardiomyocytes in vitro by direct suspension method. The expression of two kinds of myocardial specific structural proteins in differentiated cardiomyocytes gradually increases with the progression of differentiation.