BALB／c小鼠精原干细胞长期培养和移植的实验研究  

Long-term culture and transplantation of spermatogonial stem cells from BALB/c mice

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,430060 [2]武汉大学人民医院泌尿外科,430060

出　　处：《中华泌尿外科杂志》2009年第8期552-555,共4页Chinese Journal of Urology

基　　金：国家自然科学基金资助项目（30400160）

摘　　要：目的建立小鼠精原干细胞（SSC）长期培养体系，探讨SSC体外增殖分化的关键因子。方法收集出生4～6d BALB／c绿色荧光小鼠睾丸，采用改良两步消化法获得细胞悬液，3次差速贴壁去除体细胞获得富集的精原细胞，采用添加生长因子的无血清基础培养液重悬，种植到小鼠胚胎成纤维细胞饲养层上培养。基础培养液为StemPro-34SFM干细胞培养基并补充15种添加成分；生长因子为10ng／ml碱性成纤维细胞因子、20ng／m[胶质细胞源性神经营养因子和200ng／ml GDNF家族受体α1。取4～5周龄BALB／c雄性小鼠15只，腹腔注射40mg／kg的白消安建立受体模型，采用三维显微注射系统将培养的SSC移植到受体左侧睾丸精曲小管内，右侧睾丸作为自身对照；分别采用体视荧光显微镜观察和HE染色检测细胞移植后睾丸生精功能恢复情况。结果改良消化富集法消化后细胞活性〉98％，SSC富集约18．5倍。饲养层培养1～2d后细胞成对称或线形排列，细胞间可见明显的胞质桥连接。3～4d后精原细胞增殖形成典型的克隆，为边缘不清楚的团块；小鼠SSC能在该培养体系中稳定培养、传代3个月。移植后2个月，体视荧光显微镜下受体睾丸内可见明显绿色阳性克隆，HE染色证实移植的SSC在受体睾丸内克隆增殖并分化产生成熟的精子。结论成功建立了BALB／c小鼠SSC培养体系，为研究SSC增殖分化调控机制及SSC移植治疗男性不育提供了实验依据。Objective To establish a long-term culture system for mouse spermatogonial stem cells（SSCs）. Methods Testis cells from 4--6 days postpartum male transgenic BALB/c mice were collected by a modified two step enzymatic digestion method. After three differential adherence selections, the enriched germ cells were finally suspended in StemPro 34 SFM medium supplemented with other nutrients factors and plated on mouse embryonic fibroblast （MEF） feeder layer. 20 ng/ml Glial cell line-derived neurotrophic factor, 10 ng/ml basic fibroblast growth factor and 200 ng/ml GDNF-family receptor al were added to the serum-free medium to promote SSCs proliferation. Adult male BALB/c mice, 4--5 weeks old, underwent intraperitoneal injection of 40 mg/kg busulfan as recipient mice. Cultured SSCs were also injected into the seminiferous tubules of the left recipient testis through micromanipulator and right testis as self-control. Testes of recipient mice were observed by a fluores- cence stereomicroscope and HE stains at 2 months after transplantation. Results By improved digestion method, the vitality of isolated testis cells was more than 98% and the stem cells was enriched about 18.5 fold. 1-2 days after transferred to MEF feeder, the round germ cells started to proliferate and had the shape of paired or aligned undifferentiated spermatogonia connected by cytoplasmic bridges. After 3--4 days, SSCs proliferated continuously and formed typical colonies. SSCs from BALB/c mice could be cultured and passaged in a steady state for 3 months. Cryostat section through the transplanted testis showed that most of seminiferous tubules were filled with germ cells expressing EGFP. HE staining further showed clearly that seminiferous tubules contained complete spermatogenesis. Conclusions SSCs from BALB/c mice could be cultured in an improved culture system for 3 months. The culture system could facilitate understanding the regulatory mechanism that governs SSCs and might provide an opportunity for the cure of infertility.

关 键 词：精原细胞 干细胞 细胞培养技术 移植 小鼠 

分 类 号：R686[医药卫生—骨科学]