以α6-integrin和c-kit为特异性表面标志分离筛选小鼠精原干细胞的可行性  

Feasibility of spermatogonial stem cells separation with alpha 6-integrin and c-kit as specific surface makers in mice

作　　者：杜贤进[1] 张茨[1] 刘春霞[1] 王玲珑[1] 余小祥[1] 

机构地区：[1]武汉大学人民医院泌尿外科,湖北省武汉市430060

出　　处：《中国组织工程研究与临床康复》2009年第36期7034-7038,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30400160)~~

摘　　要：背景:从目前文献报道来看,精原干细胞分离效率较高且较为公认的方法是通过隐睾模型结合表面标志进行多参数筛选。目的:探讨α6-integrin和c-kit作为分离筛选精原干细胞特异性表面标志的可行性。设计、时间及地点:随机对照动物实验,于2006-05/12在武汉大学人民医院完成。材料:6周龄雄性昆明白小鼠40只,随机分为隐睾组、正常对照组,20只/组。方法:隐睾组小鼠建立隐睾模型,麻醉后腹部正中切口,将两侧睾丸拉入腹腔,将脂肪垫靠近附睾处各缝一针,分别固定在侧腹壁。正常对照组小鼠不进行任何干预。造模后两三个月,采用传统的两步消化法获取生精上皮单细胞悬液,加入FITC标记的抗α6-integrin抗体和PE标记的抗c-kit抗体,利用流式细胞仪进行α6-integrin+和c-kit-双重筛选,锥虫蓝染色监测细胞活性。主要观察指标:隐睾的形态学变化,精原干细胞分选结果。结果:隐睾小鼠的生精小管内细胞排列紊乱,层次及管腔消失,小管中央可见分裂相细胞,细胞数明显减少。与正常对照组比较,隐睾组sidescatterlow、Forwardscatterhi区的睾丸细胞增多,图形向左上移位。α6-integrin+和c-kit-细胞分布存在明显的相互偏离,即α6-integrin+细胞绝大多数不是精原干细胞,c-kit-细胞绝大多数也不是精原干细胞。从隐睾组筛选出具有sidescatterlow、α6-integrin+、c-kit-特征的细胞数占睾丸细胞总数的2.8%,此即为精原干细胞,锥虫蓝监测结果显示细胞活性达95%以上。结论:采用α6-integrin和c-kit这两种表面标志进行精原干细胞的筛选,尽管可以提高细胞悬液中的精原干细胞纯度,但均缺乏特异性。BACKGROUND： It is accepted that the best method for spermatogonial stem cells separation is using artificial cryptorchism model combined with surface makers. OBJECTIVE： To explore the feasibility of separation spermatogonial stem cells with α6-integrin and c-kit as specific surface markers. DESIGN, TIME AND SETTING： The randomized control experiment was performed at the Renmin Hospital of Wuhan University from May to December 2006. MATERIALS： Forty adult, white Kunming mice with 6 weeks old were randomly divided into cryptorchidism and control groups, with 20 animals in each group. METHODS： Artificial cryptorchidism model was prepared by made an incision at the median of abdomen, and testis was pulled into abdominal cavity, which was fixed at the each side of lateral abdominal wall. There was no treatment in the control group. The single cell suspension of seminiferous epithelium was obtained by traditional two step enzyme digestion at 2-3 months after operation. FITC-conjugated anti-α6-intergrin antibody and PE-conjugated anti-c-kit antibodies were added. Then the cells with low side scatter light-scattering properties were sorted and positively stained for α6-intergrin and negative c-kit expression. Meanwhile, the viability of the isolated cells was assessed by trypan blue staining. MAIN OUTCOME MEASURES： The morphological changes of cryptorchidism, and the sorting results of spermatogonial stem cells. RESULTS： Cell distribution in seminiferous tubule was disorder with reduced numbers. The layer and lumens were disappeared and cell division phase could be seen in the center of tubules. Compared to the control group, the testicular cells in the cryptorchidism group were increased in the side scatterlow, Forward scatterhi areas, with figure left-upward displacement. The distribution of a6-integring and c-kit cells were deviated each other, it named that most α6-integrin; cell were not spermatogonial stem cells, so do the c-kitcells. Only 2.8% of testicular cells exhibited side scatterlow, α

关 键 词：精原干细胞 细胞分选 表面标志 

分 类 号：R394.2[医药卫生—医学遗传学]