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

THE STUDY OF DIRECT DIFFERENTIATION OF MICE EMBRYONIC STEM CELLS INTO CARDIOMYOCYTES INDUCED BY TGF-β_1 AND CO-CULTURE WITH VISCERAL ENDODERM LIKE END-2 CELLS

目的添加转化生长因子-β1（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—3d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—3dEBs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。

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 cocultured 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 cardiacspecific 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 purified cells could be gained. Conclusion By using combination induction method it could achieve a synergistic effect on the differentiation of ESCs into the cardiomyocytes.