昆明小鼠胚胎干细胞向心肌细胞的分化

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。

The differentiation of Kunming mouse embryonic stem cells into myocardial-like cells

Objective To study the culture and differentiation of embryonic stem ES cells. into the myocardial-like cells in Kunming mice. Method Feeder layer was produced and embryonic stem cells of Kunming mice were cultured by icraiin. The expression of cardiac troponin T （cTnT） and cardiac-specific proteins myosin light chain （MLC-2v） was determined by immunofluorescence method. Results EBs were formed into embryoid bodies 5 days after ES cells suspension gathered, 65% of EB cells were found in beating 10 days after induction with significant difference compared with blank control group and negative control group. Some EB cells have 1-3 beating points with the rhythm of about 50-80 times/minute. The beating cardiomyocytes derived from ES cells expressed cTnT and MLC-2v positively 10 days after induction. Conclusion ES cells were successfully cultured into beating myocardial syncytial Using 10-7 mol/L icariin.