转化生长因子β-1联合内脏内胚层样END-2细胞共培养对小鼠胚胎干细胞体外分化为心肌细胞的实验研究

目的添加转化生长因子_1β(TGF_β1)以及与内脏内胚层样END_2细胞共培养,定向诱导胚胎干细胞(ESCs)分化,探索联合使用化学诱导法与共培养法对ESCs的心肌细胞定向诱导分化作用。方法将ESCs悬浮培养形成2~3 d类胚体(EBs),再向培养液内添加TGF_β1,或(和)将2~3 d EBs与END_2细胞或END_2细胞条件培养液共培养。自然分化为对照组。免疫荧光技术检测心肌细胞特异性肌动蛋白(α_actin)及肌钙蛋白T(TnT)的表达,透射电镜观察分化心肌细胞的超微结构。结果向培养液添加TGF_β1或将2~3 d EBs与END_2细胞或END_2细胞条件培养液共培养,各自有(43±2.08)%(P<0.01),(69±3.61)%(P<0.01),(65±3.06)%(P<0.01)的EBs出现自发节律性收缩,均表达心肌细胞特异性蛋白α_Actin和TnT,观察到心肌样超微结构。自然分化组发生自发节律性收缩的EBs只有(12±1.53)%,尤其是联合使用两种诱导方法,自发性收缩的EBs高达(91±1.52)%(P<0.01),收缩区域较单一诱导组大,且细胞形态较单一。结论联合使用化学诱导和共培养2种诱导法对ESCs的心肌细胞定向分化有协同作用。

THE STUDY OF DIRECT DIFFERENTIATION OF MICE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES INDUCED BY TGF-β1 AND CO-CULTURE WITH VISCERAL ENDODERM LIKE END-2 CELLS

Objective To study the differentiation of embryonic stem cells(ESCs) induced by transforming growth factor-β1(TGF-β1) and co-cultured with visceral endoderm like END-2 cells,and explore cardiomyocytes induction effects of the combined techniques Methods Day 2-3 embryoid bodies (EBs) were derived from ESCs,and then TGF-β1 was added or/and co-cultured with END-2 cells or END-2 cells conditioned medium.Spontaneous differentiation was as a control.The expression of cardiac specific α-sarcmeric actin(α-actin) and cardiac troponin-T(TnT) was detected by immunofluoresence staining.The ultrastructural analysis for ESCs-derived cardiomyocytes was scanned by transmission electron micrograph. Results The total percentage of beating EBs treated with TGF-β1, co-cultured with END-2 cells,or END-2 cell conditioned medium was(43±2.08)%,(69±3.61)%,(65±3.06)%，respectively.All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for α-actin and TnT,and could be observed the cardiac-specific ultrastructure.Interestingly,the total percentage of beating EBs treated with the combined method was (91±1.52)%.(P<0.01),and the beating areas were bigger,and more