BALB/c小鼠精原干细胞体外长期培养和鉴定  

Long-term Culture and Identification of Spermatogonial Stem Cells from BALB/c Mice in vitro

作　　者：申复进[1] 张茨[2] 杨嗣星[2] 熊云鹤[2] 廖文彪[2] 杜贤进[2] 王玲珑[2] 

机构地区：[1]武汉大学人民医院妇产科,湖北武汉430060 [2]武汉大学人民医院泌尿外科,湖北武汉430060

出　　处：《中华男科学杂志》2008年第11期977-981,共5页National Journal of Andrology

基　　金：国家自然科学基金(30400160)

摘　　要：目的：建立小鼠精原干细胞长期培养体系，探讨精原干细胞体外增殖分化的关键因子。方法：收集出生4～6dBALB／c绿色荧光小鼠睾丸，采用改良的两步消化法获得细胞悬液，种植到铺有明胶的培养皿中，分别于种植后1、5、24h通过差速贴壁法去除体细胞，获得高度纯化的精原干细胞，种植到丝裂霉素C处理的小鼠胚胎成纤维细胞饲养层上培养。基础培养液为StemPro-34SFM干细胞培养液并补充15种添加成分；添加20ng／ml大鼠胶质细胞源神经营养因子（GDNF）、10ng／ml碱性成纤维细胞生长因子（bFGF）和200ng／mlDDNF受体α1（GFRM）促进精原干细胞增殖。分别采用免疫荧光染色和RT—PCR检测精原干细胞已知抗原和标志性基因的表达。结果：饲养层上培养3～4d后精原干细胞增殖形成典型的克隆，为边缘不清楚的团块状；小鼠精原干细胞能在该培养体系中稳定传代培养达3个月。免疫荧光共聚焦显微镜观察示Oct-4特异性表达在培养的精原干细胞核上；而GFRM显著表达在培养的精原干细胞膜表面；RT—PCR也证实培养的精原干细胞表达Oct-4、GFRal、Sox2和c—Ret等象征未分化精原细胞的标志基因。结论：BALB／c小鼠精原干细胞可在体外长期培养（3个月），该培养体系的建立将为研究精原干细胞增殖分化调控机制及精原干细胞移植治疗男性不育奠定基础。Objective： To establish a long-term culture system for mouse spermatogonial stem cells （SSCs）and to discuss the key factor that supports mouse SSC self-renewal and proliferation. Methods： Testis cells from 4-6 days postpartum male transgenic BALB/c mice were collected by a modified two-step enzymatic digestion method and plated on 0.2% gelatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast （MEF） feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor （ GDNF）, 10 ng/ml basic fibroblast growth factor （bFGF） and 200 ng/ml GDNF-family receptor cd （GFRcd） were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis. Results： After 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months, Immunocytochemical staining showed that Oct-4 was specifically expressed in the cultured SSC nucleus and GFRα1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRα1, Sox2 and several other special genes resembling undifferentiated spermatogonia. Conclusion： SSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

关 键 词：精原干细胞 培养 移植 小鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]