【作者】 邹康；

【导师】 吴际；

【作者基本信息】 上海交通大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 生殖生物学的传统观点认为,雌性哺乳动物出生后生殖腺内的生殖细胞不能增殖,随着年龄的增加而逐步减少,最终全部消失而失去生育能力。然而, 2004年Johnson等经过测定小鼠不同天数未闭锁卵泡数和闭锁卵泡数,结果发现两者存在不吻合性,即卵泡闭锁的发生率要高于未闭锁卵泡数量的减少率,因此推测卵巢具有增殖活性。本研究通过定位新生和成年小鼠卵巢内增殖的生殖细胞来寻找体内存在生殖干细胞的证据,再利用生物信息学工具预测出生殖细胞标志蛋白MVH含有跨膜结构域可以用于磁珠分选,然后用胶原酶和胰蛋白酶消化卵巢和MVH磁珠分选的方法相结合进行细胞的分离纯化,最后将纯化的细胞在与精原干细胞类似的培养条件下进行体外培养。在该培养条件下,我们成功的建立了细胞系,体外培养传代达60代以上。随后,我们通过RT-PCR、免疫荧光细胞化学、Cobra等方法从mRNA水平、蛋白水平、表观遗传学水平等多个层面上鉴定该细胞的生殖干细胞特征,并且通过细胞的异体卵巢移植使不育小鼠恢复生育能力,从而证实其生殖干细胞的功能。经过体外和体内实验的验证,我们不仅确定了所分离得到的细胞为小鼠雌性生殖干细胞,而且证实所建立的小鼠雌性生殖干细胞系能够在体外长期维持。随后,我们又通过改善纯化步骤和寻找新的标志物进一步提高了小鼠雌性生殖干细胞的分离纯化效率,进而提高建立该细胞系的成功率。小鼠雌性生殖干细胞的发现和成功的体外培养建系打破了生殖生物学的传统观点,开辟了一个新的研究领域;在临床上,对妇科疾病和不育症的治疗可能带来新的突破;在医疗保健上,有可能用于研发延缓更年期和抗衰老的药物;同时也为转基因技术提供了一种新的工具,因此该研究成果有重大的理论和实际意义。更多还原

【Abstract】 In traditional view of reproductive biology the germ cells in mammal ovaries are considered lack the capacity of mitosis after birth, fertility lost when germ cells in the pool exhaust with the growth of age. However, Johnson and colleagues in 2004 found the discordance in follicle loss versus atresia: the incidence of atretic follicles was higher than the decline of the follicles, by counting the number of non-atretic and atretic follicles in mouse ovaries at different ages, which indicated the capacity of proliferation in mouse ovary. In search of the evidence of germ cell proliferation in neonatal and adult mouse ovaries, our group characterized the proliferating germ cells in vivo, predicted the existence of transmembrane domain of MVH as a sorting marker, and isolated these putative germ line stem cells by double enzymes digestion and subsequent MVH-magnetic beads cell sorting. Cultured with similar condition of spermatogonial stem cells, female germ line stem cell (FGSC) lines were established and have been maintained for more than 60 passages. Subsequently, we characterized these cells on mRNA, protein, epigenetic levels by RT-PCR, fluorescent immunocytochemistry, Cobra assays, and verified their biological function by ovary transplantation assays, finally identified them as germ line stem cells. Furthermore, we enhanced the sorting efficiency by optimizing the purification steps and using different markers. The improvement of purification protocol facilitates the establishment of FGSCs line. The discovery of FGSCs and the establishment of FGSC line modified the traditional doctrine of reproductive biology and opened up a novel field, and brought light for the cure of female infertility and gynecological diseases in clinic as well. Moreover, we developed a new tool for transgenic engineering. Therefore, our discovery has great significance on reproductive biology theory and potential for research and clinic use.更多还原