【作者】 刘靖；

【导师】 周荣家； 程汉华；

【作者基本信息】 武汉大学 ， 生物学、遗传学， 2017， 博士

【摘要】 哺乳动物雌性生殖研究领域传统观念认为,哺乳动物如小鼠和人类在出生时或者出生数天内卵巢中卵细胞的数目便确定了,卵巢中卵细胞无更新能力,不断出现的卵泡发生过程最终导致卵细胞的耗尽。本研究通过多次贴壁法从出生后雌性卵巢中分离出雌性生殖干细胞,在体外培养中成功发现生殖细胞旺盛分裂现象。证明了在ICR小鼠品系中,出生后小鼠卵巢中仍然存在着能旺盛分裂的雌性生殖干细胞。通过对培养体系中有丝分裂的统计分析,发现不同鼠龄卵巢具有的雌性生殖干细胞数目并不一致,而是随着小鼠卵巢发育逐步减少。在出生后1-3天的新生小鼠卵巢中,能进行有丝分离的雌性生殖细胞数目最多。通过观察雌性生殖细胞分裂现象,发现进行有丝分裂的雌性生殖干细胞直径大小为15-25 μm,在增殖过程中出现细胞连接现象,即分裂后的细胞核分离而细胞质仍然连接。这一现象与PGC向卵原细胞分化时形成的生殖细胞囊类似。在雌性生殖干细胞培养过程中,发现GDNF的去除有助于雌性生殖细胞的生长,当雌二醇(E2)和GDNF同时存在时,生殖细胞生长进一步受到抑制。为了研究GDNF与E2对雌性生殖细胞分化的协同促进作用,对Gfra1启动子进行了分析。通过荧光素酶报告系统,证明E2与雌激素受体蛋白ESR1正调控Gfra1基因的表达。通过相关实验认为,E2激活生殖干细胞中Gfra1基因的表达,通过GDNF-GFRA1-RET通路促进雌性生殖细胞的分化。磁珠分离法得到VASA+雌性生殖干细胞在细胞形态和表达谱与贴壁法分离的雌性生殖干细胞存在着差异。磁珠分选雌性生殖干细表达Sox2和Nanog,在培养过程中容易出现向多能性细胞转化现象。根据相关实验数据,认为磁珠分选的雌性生殖干细胞更接近原始生殖细胞状态,而贴壁法得到的雌性生殖干细胞更接近卵原细胞。本研究提出了新的雌性生殖干细胞分离方法,并对新方法得到的雌性生殖干细胞与磁珠分离法得到的雌性生殖干细胞生物学特征进行了比较。本研究丰富了对小鼠雌性生殖干细胞来源、增殖和分化的认识。更多还原

【Abstract】 It is regarded that the number of fermale germ cells in mammalian ovary has been determined at birth or soon after birth,the germ cell pool is thought to be extuasted due to the inability of fermale germ cell renewal,but this traditional idea has been challenged recently.Here I report a new method to isolate female germline stem cells(FGSC)from neonatal ovary.Through multi-round attachment in gelatin-coated wells,ovary germ cells were enriched after attachment.During short-term culture,some female germ cells exhibited dividing activity in FGSC culture condition,indicating that fermale germ cells still have self-renewal ability after birth.Moreover,maximum activity of cell dividing in the female germ cells can be obtained from ovary of newborn mice,especially postnatal 1-3 days.The diameters of germ stem cells were among 15-25 μm,and actively dividing cells finally formed germ cell cyst-like structure,in which cells contacted through cytoplasm bridge,while nucleus were sepearated.To investigate which cytokine is indispensible during cell dividing,growth factors deperived condition was used in cell culture.I observed that cells in GDNF deperived condition still exhibited diving activity.In addition,when the estrogenic hormone E2 was added in GDNF-contained culture conditon,cell proliferation was significantly depressed.These results suggested that GDNF and E2 could act coordinately to induce germ cell differentiation,and the differentiated germ cells were unable to be maintained in the culture and led to cell number decline.To testify the hypothesis I conducted Dual-Luciferase Reporter Assay to analyse the influence of E2 hormone on Gfral promoter acitivity.E2 hormone can increase Gfra1 transcriptional activity,thus led to an increasment of GDNF receptor protain GFRA1,This observation indicated that the increasement of GDNF-GFRA1-RET signaling could trigger the germ cell differentiation.When compared with muli-round attachement islated germline stem cells,the VASA+ female germline stem cells isolated through MACS method exhibited different characteristics such as cell diameter,stem related genes expression and differentiation ability,which indicated that MACS isolated germline stem cells resembled PGC,while muli-round attachement islated stem cells were more similar to oogonial cells.In conclusion,multi-round attachment method is sufficient for fermle germ cells isolation,numbers of fermale germline stem cells in ICR strain mice decrease with aging,and E2 and GDNF can influence the fermale germ cell differtiantion.These results enrich our understanding of mice fermale germ cell features.更多还原