小鼠精原干细胞的分离培养及相关标记物检测  

Isolation and Culture of Mouse Spermatogonial Stem Cells and Determination of the Related Markers

作　　者：郑艳波[1] 李毅[1] 甄永苏[1] 

机构地区：[1]中国医学科学院北京协和医学院医药生物技术研究所肿瘤室,北京100050

出　　处：《中国医学科学院学报》2013年第3期243-248,共6页Acta Academiae Medicinae Sinicae

基　　金：中央级公益性科研院所基本科研业务费专项(IMBF201006)~~

摘　　要：目的建立一套简单高效的小鼠精原干细胞(SSCs)分离及体外培养体系,检测相关干细胞标记物的表达,为将SSCs用于抗肿瘤干细胞药物的筛选及鉴定奠定基础。方法采用组织化学法比较DBA/2乳鼠(6～8天龄)和成年鼠(26～28周龄)曲细精管解剖结构的特点。选取乳鼠睾丸制备原代细胞,比较不同消化酶的消化效率。利用差异贴壁法即睾丸体细胞在明胶上贴壁而精原细胞不贴壁的特点,富集SSCs。比较以StemPro34和α-MEM为基础培养基的培养体系及明胶、血清等因素对细胞贴壁及生长性能的影响,观察不同培养时间的细胞形态,免疫荧光法鉴定SSCs及肿瘤干细胞(CSCs)相关标记物的表达。结果乳鼠睾丸中精原细胞含量相对较高,胰蛋白酶消化后能获得较多数量的原代细胞。在以StemPro34为基础培养基,含1%胎牛血清及明胶铺板的条件下体细胞贴壁较充分。分离的精原细胞在以丝裂霉素C处理过的体细胞为饲养层的环境中生长良好,呈现出典型的SSCs生长特性,13 d左右可形成明显的细胞球。免疫荧光结果显示,SSCs标记物胶质细胞源性神经营养因子(GDNF)家族受体α1(GFRα1)及VASA蛋白在细胞球中高表达;CSCs标记物CD44在As、Apr、Aal各期细胞及细胞球的内部细胞中均有表达,而体细胞中则几乎不表达。结论初步建立了DBA/2小鼠SSCs的分离及体外培养体系,CD44在发育早期的精原细胞中表达较强。Objective To establish a simple and highly effective isolation and culture system of mouse spermatogonial stem cells （SSCs） and detect the expression of stem cell-related markers in the isolated cells. Methods The structures of seminiferous tubules of neonatal （6-8 days of age） and adult （26-28 weeks） DBA/2 mice were compared using histochemical examination. Testes of neonatal mice were selected for preparing primary ceils. The digestive efficiency of different enzymes was compared. SSCs were isolated according to the different binding abilities of testicle somatic ceils and SSCs to gelatin matrix. The effects of different base culture media such as StemPro34 and α-MEM, gelatin, and serum on the SSCs binding activity and growth were studied. The cell morphology was observed during the culture process. Immunofluorescence was used to detect the expression of SSCs and cancer stem cells （CSCs） -related markers in SSCs. Results The content of SSCs in the testes of neonatal mice was relatively higher than that in adult mice. Trypsin showed the highest digestive efficiency. In StemPro34 supplemented with 1% fetal bovine serum and on the gelatin matrix, testicular somatic cells could bind with the plate efficiently. Spermatogonial cells grew well when using mitomycin C-treated testicular so- matic ceils as feeder cells and showed typical characteristic of SSCs. After 13 days of culture, spermatogonial cells formed cell clusters. Immunofluorescence assay showed that SSCs markers glial cell line-derived neurotrophic factor （GDNF） family receptor α1 （GFRα1） and VASA protein were highly expressed in the cell clusters. CSCs marker CIM4 was expressed in the As, Apt, Aal and the inner ceils of the cell clusters, while seldom expressed in the somatic ceils. Conclusions An isolation and culture system of SSCs derived from DBA/2 mice was established. CD44 is highly expressed in the early stage of spermatogonial cell development.

关 键 词：精原干细胞 分离 培养 肿瘤干细胞 CD44 

分 类 号：Q28[生物学—细胞生物学]