多基因非整合性过表达诱导山羊骨髓间充质干细胞向雄性生殖细胞分化的研究

Purpose: this study aims to describe a simple, nonintegrating strategy based on administration of synthetic mRNA for efficiently directing the differentiation of goat bone marrow mesenchymal stem cells (BMSCs) into primordial germ cells(PGC), then getting through meiosis, and differentiate into haploid male germ cells..Contents: ①Based on Bmp-4 has an important role in the process of primordial germ cells, BMSCs were induced to differentiate into PGCs by Bmp-4 gene transient expression with synthetic Bmp-4 mRNA, and the PGCs could be sorted out by the system of germ cell-specific expression of Vasa gene promoter driving the expression of green fluorescent protein; ②Based on the regulation roles of the Stra8, Dzal and Boule on male germ cell meiosis, the PGC induced from BMSCs further differentiated into haploid sperm cells by synthetic Stra8, Dzal and Boule mRNA, and the male germ cells induced from the BMSCs were then identified by cell morphology, gene and protein expression, imprinted gene methylation,the ability supporting embryo development in vitro and the differentiation capability in vivo.

目的：本项目旨在采用体外合成的mRNA转染细胞实现多个生殖细胞特异基因的非整合性过表达，更好地模拟体内生殖细胞发育分化过程，最大限度地诱导山羊骨髓间充质干细胞（BMSCs）分化为早期原始生殖细胞（PGC）并顺利通过减数分裂，形成单倍体的雄性生殖细胞。内容：①基于Bmp-4在PGC形成过程中的重要作用，通过体外合成的Bmp-4 mRNA诱导BMSCs分化为PGC，并通过Vasa-GFP早期生殖细胞的报告模型，利用流式细胞仪分选出PGC样细胞。②基于Stra8，Dazl和Boule对雄性生殖细胞减数分裂的重要调控作用，体外合成这三种基因的mRNA, 尝试通过不同基因组合诱导BMSCs分化来源的PGC启动减数分裂，形成单倍体的精子样细胞，并分别从细胞形态、基因和蛋白表达特征、印记基因甲基化情况、体外支持胚胎发育和体内分化能力等方面对诱导生成的雄性生殖细胞进行鉴定。

骨髓间充质干细胞(BMSCs)具有取材方便以及胚胎干细胞类似的多向分化潜能，是目前用于体外诱导分化为生殖细胞的研究热点。为此，我们在此课题的资助下，以山羊BMSCs为试验对象，通过体外合成多个生殖细胞特异基因的mRNA实现基因的非整合性表达，建立和优化了山羊BMSCs向雄性生殖细胞分化过程中的诱导技术体系，重点开展了下列研究：①成功建立了基于Vasa基因的早期生殖细胞绿色荧光报告模型；②利用RA和Bmp-4体外诱导Vasa-GFP转染的山羊BMSCs 向早期生殖细胞发生了分化；③ 利用Stra-8, Dazl 和Boule 的质粒和mRNA实现多基因过表达诱导山羊BMSCs 来源的早期生殖细胞启动减数分裂，向雄性生殖细胞方向发生分化；④分析了山羊BMSCs体外分化的雄性生殖细胞的特征，表现出精子状形态, 并表达减数分裂后的相关标记基因。通过这些研究，为哺乳动物雄性生殖细胞的发生及机理建立了可靠的模型，为干细胞的再生医学及畜牧产业的应用提供新的途径。