体外分化过程中小鼠拟胚体的细胞学特征及基因表达模式  

DEVELOPMENTAL CHARACTERISTICS AND GENE EXPRESSION PATTERNS OF MOUSE EMBRYONIC STEM CELLS-DERIVED EMBRYOID BODIES IN VITRO

作　　者：秦洁[1] 郭新[1] 张键荣[1] 桂耀庭[1] 蔡志明[1] 

机构地区：[1]北京大学深圳医院男性生殖医学与遗传深圳市重点实验室深圳北京大学香港科技大学医学中心,深圳518036

出　　处：《解剖学报》2008年第4期547-551,共5页Acta Anatomica Sinica

基　　金：广东省自然科学基金资助项目(7300931);广东省医学科研基金资助项目(B2007158);国家自然科学基金资助项目(30500543);"985工程"资助子项目(985-2-054-29);深圳市科技局资助项目(JH200505270413B);国家自然科学基金资助项目(30700824)

摘　　要：目的探讨小鼠胚胎干细胞来源的拟胚体(EBs)形成以及分化发育90d过程中的形态学特征及基因表达模式。方法将小鼠胚胎干细胞通过悬浮培养获得EBs,EBs在不添加其他诱导因子的体系中连续悬浮培养,采用形态学观察、免疫组织化学染色和RT-PCR检测EBs培养过程中的细胞发育分化特征和基因表达模式。结果将胚胎干细胞转移到去除白血病抑制因子(LIF)的悬浮培养液中培养24h左右即可形成EBs;培养3d左右,部分EBs出现简单胚体结构;在EBs分化7d左右,逐步向空腔化胚体发育;培养9d左右,EBs可发育为原始的囊性胚体结构;培养11d左右,EBs形成典型的、结构完整的三胚层结构。HE染色结果显示,发育早期的EBs包含大量尚未完全分化的ES细胞,随着培养时间的延长,EBs向空腔化、囊性胚体发育,逐渐形成类似于早期组织的形态。悬浮培养7周左右,EBs的发育分化基本停止。免疫组织化学染色结果表明,中胚层心肌细胞特异性标志蛋白cTnT,外胚层特异性标志蛋白Nestin在15d的EBs中表达呈阳性。RT-PCR检测也显示形成的EBs表达外胚层特征性基因Vimentin、Nestin,中胚层特征性基因Flk-1、Gata-1和内胚层特征性基因Transthyretin、-αfetoprotein。结论小鼠胚胎干细胞来源的EBs具有分化为3个胚层的能力,EBs形成的三维结构能够有效的启动细胞的分化;EBs来源的三胚层细胞的发育分化时序和特征,将为鉴定胚胎干细胞来源的分化细胞提供重要参照。Objective To explore the developmental characteristics and gene expression patterns of mouse embryonic stem cells （mESG）-derived embryoid bodies （EBs）. Methods The EBs were derived from undifferentiated mESCs by suspending culturs in leukemia inhibitory factor （LIF） free media. The morphological character istics and genes expression related to the sequential stages of embryonic development was detected by morphological observation, immunohistochemistry and RT-PCR over 90 day＇s duration. Results The primary round and packed EBs were formed within the first 24 hours of suspension culture, which also comprised many of undifferentiated mES. The simple embryonic bodies formed through 3-day of differentiation, and the EBs developed to embryonic cavity-like structures at 9 days. Subsequently, the EBs formed a typical and integral trilaminar structure at 11 days. Early stage EBs always developed to a columnar epithelium with the formation of a central cavity and was also cystic. From the seventh week on, the EBs growth ceased. At fifteen day, the EBs derived from mES expressed a trilaminar protein markers Nestin and cTnT, The expression of other genes associated embryonic development such as Vimentin, Nestin , Flk-1, Gata-1, Transthyretin and α-fetoprotein were detected by RT-PCR. Conclusion EBs can differentiate into the dimensional cell aggregates and initiate mES differentiation procedures during the long periods of culture in vitro. Our results provided a molecular and cellular basis for the identification of ES-derived functional differentiation cell types.

关 键 词：胚胎干细胞 拟胚体 发育 分化 免疫组织化学 小鼠 

分 类 号：R321.1[医药卫生—人体解剖和组织胚胎学]