生殖特异性miRNA通过Stra8对鸡ES细胞向雄性生殖细胞分化的调节机制

Male germ cells not only provide a new ideas for the treatment of male sterility and reproductive physiology research, but also supply cell models for studying the regulatory mechanism of the germ cells and understanding the generating process of sperm, more importantly, which does not involve the ethical restrictions and immune rejection problems. Although many scientists have been dedicated to study ES cells differentiation into the male germ cells in vitro, the induction system used by various research groups was quite different, and there was no a stable and effective induction system, and resulted in low induction efficiency. Some research groups have access to the ES cell-derived gamete cells, but were still a lack of functional verification, cannot meet the needs of the production and practice.Therefore, this study base on the Stra8 gene found by RNA-seq and induction in vitro, the cell differentiation model of germ cell derived ES cell was established, which was induced by RA. The expression level of miRNA and the key gene was detected during the differentiation process, the regulation mechanism of miRNA was analyzed, the best induction system will be obtained and the differentiation mechanism of germ cell was clarified to improve the induction efficiency of ES cell differentiation into male germ cell in vitro and provide theoretical supportion and practical basis to clarify the mechanism of the germ cells.

雄性生殖细胞不仅为治疗雄性不育和开展生殖生理学研究提供了新思路，并为研究生殖细胞发生调控机制和了解精子发生过程提供细胞模型，且不涉及伦理道德和免疫排斥的限制。尽管科学家已致力于ES细胞向雄性生殖细胞的诱导分化研究，但尚缺乏稳定有效的诱导体系，且诱导效率较低。虽然一些研究组已经获得ES细胞来源的配子细胞，但尚缺乏功能性的验证，不能满足生产和实践的需要。因此，本研究在前期RNA-seq和体外诱导获得调控ES细胞向雄性生殖细胞增值和分化的关键基因Stra8的基础上，建立RA诱导ES细胞向雄性生殖细胞增值和分化的细胞模型，分析雄性生殖细胞分化过程中生殖细胞特异miRNA对关键基因Stra8的调控机制，通过体内和体外试验验证miRNA的调节方式，旨在建立鸡ES细胞向雄性生殖细胞分化的体外最佳诱导模型，为提高ES细胞体外定向诱导为雄性生殖细胞的效率和阐明生殖细胞的发生机制提供理论基础。

雄性生殖细胞不仅为治疗雄性不育和开展生殖生理学研究提供了新思路，并为研究生殖细胞发生调控机制和了解精子发生过程提供细胞模型，且不涉及伦理道德和免疫排斥的限制。尽管科学家已致力于ES细胞向雄性生殖细胞的诱导分化研究，但尚缺乏稳定有效的诱导体系，且诱导效率较低。虽然一些研究组已经获得ES细胞来源的配子细胞，但尚缺乏功能性的验证，不能满足生产和实践的需要。因此，本研究在前期RNA-seq和体外诱导获得调控ES细胞向雄性生殖细胞增值和分化的关键基因Stra8的基础上，建立RA诱导ES细胞向雄性生殖细胞增值和分化的细胞模型，分析雄性生殖细胞分化过程中生殖细胞特异miRNA对关键基因Stra8的调控机制，通过体内和体外试验验证miRNA的调节方式，旨在建立鸡ES细胞向雄性生殖细胞分化的体外最佳诱导模型，为提高ES细胞体外定向诱导为雄性生殖细胞的效率和阐明生殖细胞的发生机制提供理论基础。.为了在体外建立视黄酸诱导家鸡ESC向雄性生殖细胞方向诱导分化的实验模型，选择第三代生长良好的ESC接种到以支持细胞作为饲养层的24孔板中，接种密度为10-5个细胞每孔，诱导培养基中添加不同浓度的RA，根据特异基因表达量和细胞形态变化，确定视黄酸浓度的最佳诱导浓度为10-5 mol/L；以鸡精原干细胞的cDNA为模板，通过3’RACE技术克隆Stra8基因的3’UTR， Targetscan生物在线软件预测到靶向Stra8基因的4个特异性较高的miRNA，并成功构建其相应的慢病毒载体；双荧光素酶活性检测结果显示， gga-miR-1a，gga-miR-31，gga-miR-218均可通过3’UTR序列抑制Stra8基因的表达，其中gga-miR-31抑制效果最佳；且体内外实验结果表明miR-31可有效靶向Stra8而抑制SSC进行减数分裂和阻碍精子生成。同时，本研究还开展了ESC和SSC体外多能性维持的研究，发现分别添加或者联合添加5-Azadc和TSA均可使Nanog基因的转录活性增强，可有效的通过提高Nanog基因的表达而维持鸡ESC在体外培养过程中的多能性。