【摘要】 目的:探讨小鼠胚胎干细胞(ESCs)在转化生长因子β1(TGFβ1)联合内脏内胚层END2细胞共培养诱导条件下分化为心肌细胞的特征。方法:小鼠胚胎干细胞在小鼠胚胎成纤维细胞(MEF)饲养层上增殖培养,先将ESCs悬浮培养形成23d的拟胚体(EBs),再将23dEBs种植到含有TGFβ1的24孔板中进行诱导,以及种植到铺有END2细胞饲养层的24孔板中进行诱导,培养液中同时加入TGFβ1。免疫细胞荧光技术检测心肌细胞特异性肌动蛋白(αActin)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。流式细胞仪检测EBs集落心肌细胞的平均分化率。结果:TGFβ1诱导组和TGFβ1联合内脏内胚层END2细胞共培养诱导组分别有43%,91%(P<0.05)的拟胚体出现自发节律性收缩,均表达心肌细胞特异性蛋白αActin和TnT,以及观察到心肌样超微结构,但在共培养的条件下,自发节律性收缩区域较大,且细胞形态较单一。两诱导组EBs集落心肌细胞平均分化率分别是35%,74%(P<0.05)。结论:TGFβ1联合内脏内胚层END2细胞共培养对小鼠ESCs向心肌细胞定向分化有协同作用,具有较高的诱导分化率。更多还原

【Abstract】 Objective: To analyze the differentiation features of mouse embryonic stem cells(ESCs) induced by TGF-β_ 1 and co-culture of visceral endoderm like (END-2) cell. Methods: Mouse embryonic fibroblast(MEF)feeders were used to promote the growth of ESCs and were maintained in an undifferentiated state. Embryoid bodys EBs aged 2-3 days were derived from ESCs,then were added to 24 holes panel containing TGF-β_ 1 and added to 24 holes panel covered a feeder layer of END-2 cells,containg TGF-β_ 1.The expression of cardiac specific α-sarcmeric actin( α-actin) and cardiac troponin-T(TnT) was detected by using immunofluoresence.The ultrastructural analysis of ESCs-derived cardiomyocytes was scanned by transmission electron micrograph.The percentage of EBs cluster-derived cardiomyocytes was evaluated by flow cytometry. Results: The total percentage of beating EBs treated with TGF-β_ 1 and TGF-β_ 1 combined with END-2 feeder layer was 43% and 91%(P<0.05),respectively. All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for α-actin and TnT, and the cardiac-specific ultrastructure could be observed. Interestingly, under the condition of co-culture with END-2 cell,the beating areas were bigger,and more purified cells could be gained. The percentage of EBs cluster-derived cardiomyocytes between the two induction groups was 35% and 74%(P<0.05), respectively. Conclusion: Added TGF-β_ 1 co-cultured with visceral endoderm like END-2 cell could achieve a synergistic effect on the differentiation of ESCs into the cardiomyocytes, and could get a higher induction efficiency.更多还原