生殖特异性miRNA对奶山羊雄性生殖干细胞增殖与分化调控的机理

It is well known that miRNAs(microRNAs), a type of short RNA molecules, play critical roles in cellular function, proliferation,differentiation, apoptosis, sex determination,germline stem cells in eukaryotic cells. Male germline stem cells (mGSCs) lie at the foundation of spermatogenesis, which is one of the most productive self-renewal systems in adult animals, and can speed the study of genetics, breeding of mammals, and transgenic engineering and cloning animals.However, it is still an issue that how many miRNAs specifically express in dairy goat mGSCs and how these miRNAs regulate the proliferation and differentiation of dairy goat mGSCs. So, we propose this project to investigate the specific expressing miRNAs in dairy goat mGSCs, and the function of the specific miRNAs in proliferation and differentiation of mGSCs and sperm formation in male dairy goat. Firstly, the mGSCs were purified by Magnetic-Activated Cell Sorting(MACS), and differential expression of miRNAs is selected and confirmed by a series of techniques including microRNA deep sequencing, quantitative PCR and in situ hybridization. To select the specific miRNAs of mGSCs in dairy goat, which are closely related to their proliferation and differentiation. Secondly, the effects of the specific differential expression of miRNAs in proliferation and differentiation of dairy goat mGSCs is revealed by their specific synthetic miRNA mimics simulation or transfection of their expression vector. Thirdly, target genes and related signal pathways of the specific miRNAs are analysed and screened out by bioinformatics,microarray chips.To reveal the mechanism of specific target, the specific target sites are analysed by Co-IP, dual-luciferase reporter assay system and RNAi, then the mechanism of interactions between the miRNAs and the target genes will be elucidated. The results will provide new scientific inforamtion to investigate the function of specific miRNA in proliferation and differentiation of mGSCs and sperm formation in dairy goat.

miRNA是一类微型RNA，对细胞增殖、分化、凋亡、性别决定及生殖干细胞等具有重要的调控作用。借助雄性生殖干细胞(mGSCs)可大大加快动物遗传育种及转基因克隆的研究。但在山羊及其它家畜的mGSCs中有哪些特异性miRNA，其对mGSCs是如何发挥调控作用等是急需解决的问题。本项目拟在已有的基础上，采用磁珠抗体标记法纯化出mGSCs；通过miRNA深度测序技术、结合定量PCR和原位杂交等手段，筛选、确定与奶山羊mGSCs增殖分化紧密相关的miRNA；特异miRNA的模拟物或载体转染奶山羊mGSCs，揭示其对mGSCs增殖与分化的作用；借助生物信息学、表达谱芯片分析筛选出miRNA作用的靶基因及相关信号通路，免疫共沉淀、双荧光素酶报告载体和RNA干扰等验证这些miRNA可能作用的特异靶位点，揭示其与靶基因在发挥生物学作用中的互作机制；探索miRNA调控mGSCs增殖与分化及精子发生的机理。

项目紧密按照原计划任务展开研究，取得了一系列创新性的进展，圆满完成了原计划任务。(1) 采用CD90和CD49f的免疫磁珠抗体分选技术有效的富集和纯化奶山羊雄性生殖干细胞，并对纯化后的细胞进行了标记基因系统的检测和培养鉴定。(2) 建立并获得永生化的奶山羊雄性生殖干细胞株。(3) 利用CD49f 结合免疫磁珠标记技术分选，深度测序获取奶山羊雄性生殖干细胞的miRNAs表达谱，通过与已报到的山羊乳腺、卵巢miRNA表达谱比较，确定了67个已命名的奶山羊睾丸特异性表达miRNAs和奶山羊雄性生殖干细胞特异性表达miRNAs。其中miR-34c、miR-449a可能与奶山羊精子发生关系密切。(4) 证实miR-34c通过靶向Stra8、Nanos2及PPARγ调控奶山羊雄性生殖干细胞的自我更新与分化。同时利用雄性生殖干细胞和奶山羊雄性生殖干细胞作为模型深入探索关键基因与miRNA调控互作机理，发现：miR-204-Sirt1调控轴调控奶山羊精原干细胞自我更新和增殖凋亡；miR-544靶向PLZF调控奶山羊雄性生殖干细胞的自我更新； PLZF通过靶向抑制miR-146a促进CXCR4表达调控奶山羊精原干细胞增殖。 . 本项目为人类和动物生殖干细胞的进一步深入研究积累了丰富的资料和奠定了坚实的技术基础，具有重要的科学意义和巨大的应用潜力。