小鼠胚胎干细胞分化形成拟胚体过程中的细胞程序性死亡

为了检测小鼠胚胎干细胞 (embryonicstemcell ,ES细胞 )体外分化的拟胚体 (embryoidbodies ,EBs)形成过程中细胞程序性死亡 (programmedcelldeath ,PCD)的发生 ,通过悬滴、悬浮培养技术定向诱导未分化的ES细胞分化为拟胚体 ,并用RT PCR检测原始内胚层、原始外胚层、中胚层、内脏内胚层 4种分子标记物在EBs中的表达 .通过TUNEL染色、电镜、激光共聚焦显微镜及Western印迹以确定凋亡发生 .结果表明 :ES细胞体外分化为拟胚体并且表达各胚层相应的分子标记物 ;在拟胚体的发育过程中出现明显的空腔化过程 ,TUNEL染色及电镜观察到凋亡生成 ,同时线粒体膜电位 (ΔΨm)在拟胚体发育过程中降低 ,通过Western印迹检测到caspase3、caspase8的激活 .表明小鼠ES细胞所分化的拟胚体可以作为研究早期胚胎发育的实验模型 ,线粒体在拟胚体的细胞程序性死亡过程中发挥重要的作用 .为进一步利用拟胚体研究细胞程序性死亡及相关信号分子在小鼠胚胎发育早期的作用奠定了基础

Programmed Cell Death of Embryoid Body During the Differentiation of Mouse Embryonic Stem Cells

To detect the programmed cell death (PCD) in embryoid bodies (EBs) derived from mouse embryonic stem cell differentiation in vitro.Undifferentiated ES cells were incubated and differentiate into EBs by hanging drop and suspension culture method. The expression of the primitive endoderm, primitive ectoderm, mesoderm, and visceral endoderm molecules was examined in embryoid bodies by RT-PCR. Apoptotic was confirmed by TUNEL staining,electro microscopy,confocal microscopy,and Western blotting.Four molecular markers were expressed in the EBs.The cavitation and PCD were detected in the development of EBs by TUNEL staining and electron microscopy.Membrane Potential (ΔΨ m) was decreased,and the cleaved-caspases3 as well as cleaved-caspase8 were activated during the development of EBs.These results demonstrated that EBs could be used to study the development of early embryo and that mitochondria play an important role in the PCD of EBs.The system of mouse ES cells differentiated into EBs in vitro provided genetic evidence to investigate PCD and relevant signaling molecules in the early developmental statge of mouse embryo.