牛成纤维细胞来源ips细胞向雄性生殖细胞诱导分化的机理研究

The exploration of in vitro inducing male germ cells via iPS technology has a profound application prospect in no matter human reproductive field or animal breeding and the preservation of endangered species. Currently the regulating mechanism of spermatogenesis maintains uncertain, which could not cater to the demands of research and clinic for the sake of a low in vitro induction efficiency of male germ, a tough repeatability and a reality of unreachable functional male gametes obtaining. This study aims to analysize out the key male germ cell-related.regulatory factors during its differentiation and the difference of relevant DNA methylation by high throughput sequencing and chip-seq technologies, then establishing a new model of sperm induction via bovine fibroblast-original pluripotent cells. This research provides a new theoretical basis of differentiation in male germ cell, opens an alternative approach to develop gene therapeutic agents, gives abundant genetic materials for livestock husbandry development, which benefits the sustainable industry enormously.

iPS技术在体外诱导雄性生殖细胞方面的探究，无论在人类生殖医学领域，还是在动物育种以及保存濒危动物资源方面均具有深远的应用前景。因调控雄性生殖细胞分化的机理仍不明确，导致体外诱导效率低、可重复性差，难以获得功能齐全的生殖细胞，满足不了研究和临床需要。本研究利用新一代的高通量测序技术和chip-seq技术，深度分析出体内精子发生以及iPS细胞向精子分化不同阶段的特异表达的转录因子和DNA甲基化的差异，系统挖掘出多能干细胞定向分化为精子细胞的关键调控因子，并在此基础上进行体外遗传操控，通过转基因技术建立高效的iPS细胞分化为雄性生殖细胞的新模型。本项目的实施为阐明雄性生殖细胞分化机制提供新的理论依据，为提高iPS细胞向雄性配子诱导效率的基础研究提供新手段，为临床新型基因药物开发提供新思路，为提高畜牧业发展和畜禽遗传资源保存提供新材料，对于促进我国畜牧业的健康可持续发展以具有深远的科学意义。

将iPS细胞诱导成生殖细胞具有重要的应用价值。尽管人们已经能够将iPS诱导形成具有功能的生殖细胞，但由于其调控机理尚不明确，不足以满足研究和临床需要。本项目首先建立了稳定的牛iPS培养体系，并成功向精子细胞诱导分化。利用新一代高通量测序技术和ChIP-seq技术，系统分析了精子发生过程中那个是四种不同类型细胞的DNA甲基化变化及之间的差异，筛选得到与精子发生相关的差异甲基化基因，并获得了参与精子发生过程的重要信号通路。最后以牛睾丸间充质干细胞为研究对象，结合本项目获取的差异基因，在体外成功诱导其向功能性生殖细胞分化。