基于鸡ESCs分化为雄性生殖细胞调控关键基因和信号通路筛选诱导剂的研究

Spermatogonial stem cells could be used to replace embryonic stem cells for disease therapy and genetic modification since it has the merits of pluripotency and do not involve in ethical and immune rejection problem, therefore, it caused great concern in the biological community. Although the scientists has performed the research about the stem differentiation for solving the problem how does abtain a large number of spermatogonial stem cells in vitro and achieved breakthrough, it is still unclear about the regulation mechanism of cells induced differentiation: how does the inducer interact with the key regulation gene, how does the germ cell differentiate into the specific cell types and what kind of inducing strategy was the most optimal. All these problem resulted in the inefficient induction, therefore, it is very hard to obtain fully function germ cells in vitro and could not meet the requirements of research and practice. The purpose of this project is to screen the key genes and signaling pathways involved in the germ cells differentiation by gene expression spectrum and immunohistochemical, verify its role of molecular regulation mechanism. Base on its molecular mechanism, the critical transcription factor and promoter related to this pathway was used to establish the cells screening model by Luciferase reporter gene and Mammalian one-hybrid, then the inducer was achieved following the above model. A suitable induction system was established by the different combination of inducer and the adding way, in order to provide a theoretical basis for clarifying the differentiation mechanism of germ cells, and supply a strong practical technical guidance for improving the inducing efficiency and clinical drug screening.

精原干细胞的可塑性使其能替代ESCs用于治疗和遗传修饰，而不涉及伦理和免疫排斥问题，故引起生物学界极大关注。然怎样能大量获得该类细胞？科学家们为此进行了干细胞定向诱导分化研究，虽取得了突破性进展，但因诱导方案迥异，生殖细胞分化机制及关键基因与诱导剂互作调控细胞分化的机理仍不清楚，导致诱导重复性差和效率低，体外难以获得功能齐全的生殖细胞，满足不了研究和实践需要。本项目旨在利用鸡基因表达谱芯片和免疫组化分析技术，筛选参与雄性生殖细胞分化的关键基因和信号通路，并探究其分子作用机理；在此基础上依据关键基因和信号通路关键分子的启动子及其转录因子，利用荧光素酶报告基因系统和哺乳动物细胞单杂交技术建立细胞筛选模型，对诱导剂进行筛选；比较候选诱导剂不同添加方式和不同添加组合的作用效果，建立适宜的诱导体系，为弄清生殖细胞分化机制提供理论依据，为提高细胞诱导效率及医学临床药物筛选建立细胞模型提供实践技术支撑。

通过RNA-Seq和Microarray对雄性ESCs，PGCs和SSCs进行高通量测序并筛选差异基因，根据GO分析和KEGG pathway富集结果寻找鸡雄性生殖细胞分化过程中关键基因和信号通路；比较不同诱导剂及诱导剂组合体外诱导ESCs生成SSCs效率，建立体外诱导ESCs生成SSCs模型：在体外诱导模型基础上，研究高通量测序过程中的关键基因和关键信号通路功能。结果：①提取雄性ESCs，PGCs和SSCs RNA，质量合格，能够进行转录组测序；②分析RNA-seq测序过程中的差异表达基因，筛选出173个与生殖分化相关的候选基因和关键信号通路18条；③分析Microarray测序过程的差异表达基因，筛选出34调关键信号通路；④采用qRT-PCR和DNA Microarray对RNA-Seq结果进行验证，三者结果一致；⑤利用不同诱导剂体外诱导ESCs分化，RA与支持细胞组，BMP4组能够体外诱导ESCs形成SSCs；睾酮和FSH则没有明显的诱导效果，但是对RA与支持细胞组有促进作用；⑥采用qRT-PCR验证RNA-seq候选差异基因SHISA2、SIX1等在诱导过程中的变化，差异基因在体外和体内变化保持一致；⑦构建DAZL-pEGFP-N1载体，定位DAZL基因在细胞核中表达；构建pcDNA-DAZL载体后转染ESCs能够促进SSCs样细胞形成；构建DAZL基因启动子不同长度的缺失载体，发现DAZL启动子核心区域在-147- -334之间并受到RA、ATRA、FSH等诱导剂诱导；构建pEGFP-C1-Stra8载体，定位Star8基因在细胞质中表达；构建pcDNA3.1- Stra8载体，体外能够促进RA体外诱导体系形成SSCs；构建Star8启动子的不同长度缺失片段，发现Star8启动子的核心区域位于-554bp～-255bp之间，并受TSA和Am80诱导剂诱导；构建pEGFP-C1-CVH，定位CVH在整个细胞中表达；⑧通过构建相应通路的关键基因的发卡载体或者使用特异抑制剂证明：视黄酸通路，HH信号通路，P450代谢通路，WNT信号，TGF-β信号，Notch信号，甾类激素合成通路，PPAR-RXRG信号在鸡雄性生殖细胞分化过程中起重要作用。