一种简便的小鼠精原干细胞分离培养方法  

A Simple and Convenient Method of Mouse Spermatogonial Stem Cells Isolation and Culture

作　　者：宋锐[1] 曹鸿国[1] 陶勇[1] 张运海[1] 刘亚[1] 黄伟玲[1] 孙雪萍[1] 殷慧群[1] 章孝荣[1] 

机构地区：[1]安徽农业大学动物科技学院安徽省地方畜禽遗传资源保护与生物育种省级重点实验室,合肥230036

出　　处：《中国细胞生物学学报》2010年第2期285-290,共6页Chinese Journal of Cell Biology

基　　金：国家高技术研究发展计划(863计划)(No.2008AA101003);安徽农业大学资助引进与稳定人才科研启动项目(No.yj2007-10)~~

摘　　要：采用一步酶消化法分离小鼠精原干细胞,比较α-MEM、DMEM培养基对体外培养的精原干细胞生长状态的影响,对精原干细胞集落进行形态观察、碱性磷酸酶(alkaline phosphatase,AKP)染色和免疫组化鉴定,并诱导精原干细胞向精子细胞分化。结果显示,以小鼠胚胎成纤维细胞作为饲养层,用α-MEM培养的精原干细胞集落较大且呈葡萄串状或念珠状,细胞状态较好;小鼠精原干细胞集落的AKP染色阳性呈紫红色;在红色荧光下精原干细胞集落的Oct-4核蛋白表达为阳性、膜蛋白c-Kit、β_1-integrin和Gfrα-1表达为阳性;精原干细胞经维甲酸(all-trans-retinoic-acid,RA)诱导可初步分化成精子样细胞。因此,采用一步酶消化法能够分离小鼠精原干细胞,α-MEM更适合小鼠精原干细胞体外培养。We isolated mouse spermatogonial stem cells （mSSCs） by the method of one-step enzymatic digestion, cultured mSSCs in vitro and compared the effect of α-MEM and DMEM on growth condition. The mSSCs colonies were identified by morphology observation, alkaline phosphatase （AKP） staining, immunohistochemistry and we induced the mSSCs differentiated into sperm-like cells. The results showed that using the mouse embryonic fibroblast cell as the feeder cell. the mSSCs colonies cultured by α-MEM were in good state and the graped-like or rosary-like; AKP staining showed the colour of mulberry as the positive; under the red fluorescent, the Oct-4 neucleoprotein of mSSCs colonies cells expressed positive and the membrane protein of C-Kit, α-integrin and Gfrα-1 expressed positive; the mSSCs treated with retinoic acid （RA） can be induced into the initial status of sperm-like cell. It was concluded that used the method of one-step enzymatic digestion could isolate mSSCs successfully and α-MEM was more suitable for mSSCs in vitro culture.

关 键 词：小鼠 精原于细胞 分离 培养 

分 类 号：S865.13[农业科学—野生动物驯养] Q813[农业科学—畜牧兽医]