人羊水干细胞诱导为卵母样生殖细胞的研究

发布时间：2019-04-24 01:28

【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。
[Abstract]:Differentiation of adult stem cells into oocyte-like germ cells will promote the study of oogenesis. According to the report, amniotic fluid stem cells have the ability to spontaneously differentiate into three embryoid types including ectoderm, mesoderm and endoderm in vitro. However, whether amniotic fluid stem cells have the potential to develop into germ cells has not been confirmed. In this study, we found that a human amniotic fluid stem cell line was isolated from human amniotic fluid 20 and 38 weeks before delivery, and could differentiate into oocyte-like cells under certain induction conditions, and expressed a variety of germ cell-related marker genes. In this study, we also found that a small number of cells formed in the process of differentiation can be further developed into more mature reproductive-like cells. A amniotic fluid stem cell line was isolated and purified from amniotic fluid of second trimester by limited dilution method. Chromosome karyotype analysis showed that it was a XXtype chromosome, and the specific marker gene of the cell line was further identified. In order to better observe the differentiation and proliferation of induced cells in vitro, we constructed the pBMP15-EGFP vector in the previous work. When human amniotic fluid stem cells were induced in conditioned medium supplemented with porcine follicular fluid, some cells with distinct morphology were observed, surrounded by a halo similar to a hyaline band. During induction, amniotic fluid stem cells could aggregate to form a large suspended three-dimensional embryoid structure, and these embryoid bodies were strongly positive for alkaline phosphatase staining. In addition, many germ cell-related marker genes, such as Oct4, Dazl, Stella, GDF9, CD117, SCP3, ZP1, ZP2 and ZP3 genes, are expressed in these cells during differentiation. When the constructed pBMP15-EGFP vector was transfected, these cells further differentiated into green fluorescent protein-positive oocyte-like cells. After further induction of amniotic fluid stem cells, we found that some of the circular cells could spontaneously differentiate into parthenogenetic blastocysts with a diameter of about 50-60 渭 m. Studies on the induction and differentiation of adult stem cells into reproductive-like cells, especially in the field of human amniotic fluid stem cells, may provide an in vitro research model to solve some problems in the early development of germ cells.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

【相似文献】