【作者】 陈帅；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 动物生物技术， 2010， 硕士

【摘要】 将成体干细胞定向诱导分化为卵母样生殖细胞,将会促进卵子发生的相关研究。根据报道,羊水干细胞具有在体外自发分化为包括外胚层、中胚层和内胚层的三个胚层类型细胞的能力。然而,羊水干细胞是否具有发育成生殖细胞的潜能尚未得到证实。在本研究中,我们发现从产前20-38周的人羊水中分离得到一株人羊水干细胞系,在一定的诱导条件下可以分化为卵母样细胞,并且表达多种生殖细胞相关的标记基因。本研究还发现分化过程中形成的少量形态明显特殊的细胞,可进一步发育到更加成熟的生殖样细胞。实验中采用有限稀释法,从孕中期的羊水中分离纯化了一株羊水干细胞系。染色体核型分析的结果显示其表现为XX型染色体,随后又进一步对该细胞系的特异标志基因进行了鉴定。为了更好地观察诱导细胞在体外的分化及增殖过程,我们在前期工作中构建了pBMP15-EGFP载体。当在添加了猪卵泡液的条件培养基中诱导人羊水干细胞时,我们观察到了一些形态明显特殊的细胞,外面包围着类似透明带样的光环。诱导过程中羊水干细胞可以聚集形成体积较大的悬浮的三维类胚体结构,并且这些类胚体碱性磷酸酶染色呈强阳性。此外在分化过程中,这些细胞还表达很多生殖细胞相关的标志基因,例如Oct4, Dazl, Stella, GDF9, CD117, SCP3 , ZP1, ZP2以及ZP3基因。当转染构建的pBMP15 -EGFP载体时,这些细胞进一步分化成绿色荧光蛋白阳性的卵母样细胞。对羊水干细胞进行进一步诱导后,我们发现其中的一些圆形细胞可以自发分化成类似孤雌囊胚的结构,直径大约在50 - 60μm左右。将成体干细胞诱导分化为生殖样细胞的研究,尤其是在人羊水干细胞领域进行的相关探索,或许可以提供一个体外的研究模型,来解决早期生殖细胞发育形成中的一些问题。更多还原

【Abstract】 Derivation of oocyte like cells from adult stem cells would benefit the study of oogenesis. It has been kwon that Human amniotic fluid stem cells (hAFS) are able to differentiate spontaneously in vitro into various somatic cell types. However, the germ cell potential of hAFS cells has never been tested. Here, we report that hAFS cells isolated from the 20 to 38 weeks’gestation have the intrinsic ability to differentiate into oocyte like cells based on several characters specially. The study also tested the hypothesis that a morphologically distinct population of cells derived from hAFS can differentiate further into more mature gametes.A XX hAFS cell line was isolated from human amniotic fluid of the 2nd trimester of gestation at the single-cell level by limiting dilution, and the specific cell surface antigens of the hAFS cells were characterized. In order to track and identify specific stages of female gamete induction, we construct a pBMP15-EGFP vector. When differentiation was induced in conditioned medium supplemented with porcine follicular fluid, we observed a subpopulation of morphologically round cells with zona pellucida-like morphology were observed which built large embryoid bodies in suspension culture. These 3D embryoid bodies were strongly alkaline phosphatase positive. During differentiation, these cells also express OCT4, Dazl, Stella, GDF9, CD117, SCP3, ZP1, ZP2 and ZP3, which are markers indicative of germ cell formation. When labeled with a pBMP15-EGFP reporter, these cells further differentiate into round GFP-positive oocyte-like cells. Some of these round cells spontaneously developed into parthenogenetic embryo-like structures, which were about 50 - 60μm in diameter.The potential of generate germ cell precursors from adult stem cells, especially hAFS cells, may provide an in vitro model to study some of the unanswered questions surrounding early germ cell formation.更多还原