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目的饲养层制作及复苏培养昆明小鼠胚胎干细胞,探索体外诱导胚胎干细胞向心肌细胞分化。方法饲养层制作,复苏及体外培养胚胎干细胞,采用一步法消化贴壁胚胎干细胞,悬浮培养5d,形成拟胚体(EB),待EB贴壁后,加入淫羊藿苷(Icraiin,ICA)诱导液对其诱导并每天观察,免疫荧光检测心肌细胞特异性肌钙蛋白T(cTnT)和心室肌球蛋白轻链(MLC-2v)的表达。结果胚胎干细胞悬浮聚集5d,形成类似球状的拟胚体,将拟胚体贴壁诱导8d,发现拟胚体中出现跳动,诱导后10d拟胚体跳动率达65%,显著高于空白对照组和阴性对照组,一个分化拟胚体中一般出现1-3个跳动点,节律约为50-80times/min,在诱导10d时跳动的合胞体,免疫荧光表明cTnT和MLC-2v表达为阳性。结论胚胎干细胞经悬浮聚集培养5d后经10-7mol/L淫羊藿苷诱导,得到了可以跳动的心肌细胞团。
Objective To culture and revitalize the Kunming mouse embryonic stem cells and explore the differentiation of embryonic stem cells to cardiomyocytes in vitro. Methods The embryonic stem cells were cultured, resuscitated and cultured in vitro. The adherent embryonic stem cells were digested with one-step method and cultured in suspension for 5 days to form embryoid bodies (EBs). After being attached to EB, Icraiin (ICA) The cells were induced and observed daily. The expression of cardiac troponin T (cTnT) and ventricular myosin light chain (MLC-2v) were detected by immunofluorescence. RESULTS: Embryonic stem cells (ESCs) accumulated in suspension for 5 days and formed a globular embryoid-like body. The embryoid body was adherently induced for 8 days and found to be beating in embryoid bodies. The bounce rate of embryoid bodies reached 65% at 10 days after induction, which was significantly higher than that of the blank control group and In the negative control group, one to three beating points generally appeared in a differentiated embryoid body body, the rhythm was about 50-80times / min, and the syncytium was beating 10 days after induction. Immunofluorescence showed that cTnT and MLC-2v were positive. Conclusion After embryonic stem cells were cultured in suspension for 5 days and induced with 10-7 mol / L icariin, the cardiac muscle cell mass that could be beating was obtained.