内脏内胚层样END-2细胞体外诱导鼠胚胎干细胞分化为心肌细胞的实验研究  

Visceral endoderm-like END-2 cell induces differentiation of mouse embryonic stem cell into cardiomyocytes

作　　者：曾彬[1] 林国生[1] 郑和忠[2] 蔡军[1] 罗浩[1] 

机构地区：[1]武汉大学人民医院心血管内科,湖北武汉430060 [2]遵义医学院附属医院神经内科,贵州遵义563003

出　　处：《心脏杂志》2006年第4期396-399,共4页Chinese Heart Journal

摘　　要：目的 探讨内脏内胚层样END-2细胞体外诱导胚胎干细胞（embryonic stem cells，ESCs）分化为心肌细胞的特征。方法 用鼠胚胎成纤维细胞（mouse embryonic fibroblasts，MEF）作为饲养层促进ESCs增殖并抑制其分化，先将ESCs悬浮培养形成2～3d的拟胚体（embryoid bodies，EBs），再和END-2细胞共培养诱导向心肌细胞分化。实验分4组。第1，2组EBs分别和END-2细胞或END-2细胞条件培养液共培养；第3组EBs和表面铺有一层琼脂糖的END-2细胞共培养；第4组自然分化组为对照组。相差显微镜下观察分化细胞的形态学变化，免疫细胞荧光技术检测心肌细胞特异性肌钙蛋白T（TnT）的表达；透射电镜观察分化心肌细胞的超微结构。结果 各实验组均可见自发节律性收缩的拟胚体。随着培养的延长。自发节律性收缩的拟胚体数目也增加，均表达心肌细胞特异性蛋白TnT，以及观察到心肌样超微结构。在和END-2细胞直接接触的诱导条件下，分化的细胞形态较单一。结论 END-2细胞通过分泌可溶性细胞因子可诱导ESCs向心肌细胞分化，直接接触在END-2细胞诱导作用中并不是必要的。但可诱导出较单一的细胞。AIM To analyze the differentiation features of embryonic stem cells （ESCs） induced by visceral endoderm-like END-2 cell. METHODS Mouse embryonic fibroblasts （MEF）were used to promote the growth of ESCs and keep them in an undifferentiated state. Day 2-3 embryoid bodies （EBs） were derived from ESCs and then co-cultured with END-2 cells to induce into cardiomyocytes. ENs in group 1 and group 2 were co-cultured respectively with END-2 cells and END-2 cells conditioned medium, those in group 3 were co-cultured with END-2 cell lain by a layer of agar. Spontaneous differentiation group was used as control. The expression of cardiac specific cardiac troponin-T （TnT） was detected by using immunofluoresence and the uhrastructural analysis of ESCs-derived cardiomyocytes was scanned by transmission electron micrograph. RESULTS EBs in all the groups displayed spontaneously beating loci. With the passage of time, the total number of spontaneously beating EBs increased. All the beating cardiomyocytes derived from ESCs expressed cardiac-specific proteins for TnT and the cardiac-specific ultrastructure could be observed. Under the condition of direct contact with END-2 cells, more purified cells could be obtained. CONCLUSION A differentiation-inducing factor secreted by END-2 cells can induce ESCs into cardiomyocytes. Direct contact between END-2 cells and ESCs is not necessarily required for the differentiation but can produce more purified cells.

关 键 词：END-2细胞 胚胎干细胞 心肌细胞 共培养 分化 

分 类 号：Q813.1[生物学—生物工程]