成年昆明小鼠雄性生殖干细胞的分离培养及其多能性研究  

Isolation and cultivation of pluripotent male germline stem cells(mGSCs) derived from adult KM mouse testis

作　　者：潘少辉[1] 胡玥[1] 张姗姗[1] 王静 李飞飞[1] 高志敏[1] 杨春荣[1] 华进联[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,陕西省农业分子生物学重点实验室,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2010年第6期23-30,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30972097);教育部重点科研项目(109148);中国博士后科学基金项目(200801438)

摘　　要：【目的】从成年昆明小鼠睾丸组织中分离具有多能性的雄性生殖干细胞(Male germline stem cells,mGSCs)。【方法】采用机械法和2步酶消化法分离出成年昆明小鼠的精原细胞,经差速贴壁后,培养在小鼠胎儿成纤维细胞(MEF)饲养层上,2周后得到类胚胎干细胞(ESCs)样mGSCs。对得到的mGSCs进行碱性磷酸酶(AP)染色、免疫荧光染色、RT-PCR、体内分化等检测。【结果】mGSCs具有类似ESCs的形态和多能性,AP染色呈阳性,免疫荧光Oct4、Vasa染色呈阳性,RT-PCR检测其表达Oct4、Nanog、Sox2等多能性基因和Vasa、C-kit等生殖细胞的标记基因,在免疫缺陷鼠体内能形成畸胎瘤,组织学检测其可形成包括3个胚层在内的多种类型细胞。【结论】从成年昆明小鼠睾丸分离的精原细胞,在体外培养去分化形成了具有类ESCs生物学特性的mGSCs,mGSCs既具有多能性,也具有生殖细胞的基本生物学特性。【Objective】 Male germline stem cells（mGSCs） were obtained from adult KM mouse testis.【Method】 Spermatogonial cells were isolated from adult mouse testis using mechanical and two-step enzymatic digestion method,and then cultured on mouse embryonic fibroblasts（MEF） feeder after differential plating.Embryonic stem cells（ESCs）-like mGSCs were obtained after 2 weeks.The characteristics of mGSCs were analyzed by alkaline phosphatase（AP）,immunofluorescence,RT-PCR and in vivo differentiation.【Result】 mGSCs was similar to ESCs in morphology and pluripotency.AP staining is positive.They expressed pluripotent genes：Oct4,Nanog,Sox2 Sand some germ cell markers such as Vasa,C-kit analyzed by RT-PCR and immunofluorescence.The transplanted cells formed mature teratomas containg derivatives of three embryonic germ layer cells when the mGSCs were injected into severe combined immunodeficiency mice（SCID）mice.【Conclusion】 These results demonstrated that isolated adult mouse spermatogonia was reprogrammed to form pluripotent ES-like mGSCs in vitro,and these cells also shared characteristics of germ cells.This will provide unlimited resource to understand their biological characteristics and clinical application.

关 键 词：成年昆明小鼠 精原细胞 雄性生殖干细胞 多能性 

分 类 号：Q813.11[生物学—生物工程] Q492.4