恒河猴精原干细胞的筛选、培养与鉴定

Selection culture and identification of rhesus spermatogonial stem cells

目的:建立恒河猴精原干细胞的筛选和培养方法。方法:采用改良的二步酶消化法分离恒河猴睾丸细胞,用改进的差异贴壁筛选法纯化恒河猴精原干细胞,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无血清培养基和小鼠胚胎成纤维细胞饲养层培养恒河猴精原干细胞,并通过形态观察、标志基因的免疫细胞化学分析和半定量RT-PCR分析鉴定培养细胞的干细胞活性。结果:分离纯化的恒河猴精原干细胞在添加了3种生长因子的培养基中能形成较大的干细胞克隆,并表达精原干细胞的标志基因。结论:本研究初步建立了恒河猴精原干细胞的培养体系,为进一步开展相关研究奠定基础。

Objective ： To establish a system of screening and culture of rhesus spermatogonial stem cells （SSCs）. Methods ： The modified two - step enzymatic digestion and the modified differential adherence selection were used to prepare rhesus testis cells and purify rhesus SSCs respectively, and the serum -free medium DMEM/F12 supplemented with glial cell line -de- rived neurotrophic factor （GDNF） , soluble GFRα1 and bFGF was used to culture the highly enriched SSCs on mouse embry- onic fibroblast （MEF） feeder layer. The activity of SSCs was examined morphologically and by immunocytochemical analysis and semi - quantitative RT - PCR analysis for marker gene expressions. Results ： The isolated ,nd highly purified rhesus SSCs could produce big colonies of SSCs in vitro and express the marker genes of Oct4, c - Ret, GFRα1 Conclusion： The prelimi- nary culture system of rhesus SSCs was established.