大鼠精原干细胞的筛选与培养  

Screening and culture of rat spermatogonial stem cells

作　　者：王庆忠[1] 王东方[1] 刘慧莲[1] 冯道俊[1] 

机构地区：[1]山东省高校生物化学与分子生物学重点实验室(潍坊学院生物与农业工程学院),山东潍坊261061

出　　处：《细胞与分子免疫学杂志》2012年第7期747-751,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：山东省自然科学基金(ZR2010CL015);潍坊市科技发展计划(2011022;20111023;2010143);潍坊学院优秀学术团队项目(2010Z03)

摘　　要：目的:建立大鼠精原干细胞的筛选和培养的方法体系。方法:采用改良的二步酶消化法分离大鼠睾丸细胞,用改进的差异贴壁分选法筛选大鼠精原干细胞,用添加了胶质细胞源神经营养因子(GDNF)、可溶性GFRα1和bFGF的DMEM/F12无血清培养基和大鼠胚胎成纤维细胞饲养层培养高度富集的大鼠精原干细胞,通过形态观察、标志基因的RT-PCR检测和免疫细胞化学分析鉴定培养细胞的干细胞活性。结果:我们的改良二步酶消化法和差异贴壁分选法能有效的分离和筛选大鼠精原干细胞,这些高度富集的精原干细胞能够存活20 d以上,形成较大的干细胞克隆,并表达精原干细胞的标志基因和具有干细胞活性。结论:成功建立了大鼠精原干细胞的分离、筛选和培养体系。AIM： To establish a system of screening and culture of rat spermatogonial stem cells （SSCs）. METHODS： We prepared the rat testis cells using the improved two-step enzymatic digestion, and then isolated the rat SSCs by the differential adherence selection method. The highly enriched SSCs were cultured in the serum-free culture medium of DMEM/FI2 supplemented with glial cell line-derived neurotrophic factor （GDNF）, soluble GFRal and basic fibroblast growth factor （bFGF） and on rat em- bryonic fibroblast （REF）feeder layer. The activity of stem cells was examined morphologically and by RT-PCR and immunocytochemical analysis for the SSCs marker gene expressions. RESULTS： The modified two-step enzymatic digestion could effectively isolate the rat testis cells, and from the isolated testis cells, rat SSCs could be successfully purified by the improved method of differential adherence selection. After over-20-day culture of the rat SSCs in ser- um-free medium, big colonies of SSCs were observed, and the activity of SSCs got affirmed by the positive expressions of SSCs marker genes. CONCLUSION： The system of screening and culture of rat SSCs has been established, which provide a basis for the long-term culture of rat and other animal SSCs.

关 键 词：精原干细胞 分离纯化 无血清培养 大鼠 

分 类 号：Q492[生物学—生理学]