鸡精原干细胞分离培养的影响因素  

Effect of different isolation methods and systems on spermatogonial stem cells from chicken

作　　者：陈昊[1] 余飞[2] 赵文明 吴信生[2] 徐琪[2] 李碧春[2] 

机构地区：[1]苏州大学医学院基础医学系,苏州215006 [2]扬州大学动物科学与技术学院,扬州225009 [3]不详

出　　处：《解剖学杂志》2008年第3期310-315,共6页Chinese Journal of Anatomy

基　　金：国家自然科学基金(30371031);高等学校博士学科点专项科研基金(20061117004)

摘　　要：目的:比较不同分离方法和培养体系对鸡精原干细胞(SSCs)生长的影响。方法:采用3种分离方法、6种培养体系对SSCs进行分离培养。结果:胶原酶+胰蛋白酶组合消化睾丸后获得的平均活细胞率显著地高于其余两种方法。在有和无饲养层细胞存在的条件下,SSCs在DMEM培养液中存活的时间分别为6.5 d和45.5 d,显著地长于在TCM199和RRMI1640中。在6种培养体系中,SSCs在有饲养层体系Ⅳ中存活时间和碱性磷酸酶(AKP)阳性克隆率分别为(45.5±3.2)d和31%±16%,显著地高于其他5种培养体系。SSCs在有饲养层体系Ⅳ中传代传至1、2和3代时,AKP阳性克隆率分别为31.6%、20%和18%。SSCs形成的克隆,经SSEA-1免疫染色,均为阳性。传代至第3代的SSCs,其染色体组型保持不变。结论:采用二酶组合法分离获取SSCs,而以DMEM为基础液形成的有饲养层体系Ⅳ较为适宜。Objective： To compare the effect of isolation methods and culture systems on spermatogonial stem cells （SSCs） from chicken. Methods： Three methods and six systems were used for isolation and culture. Results： The results showed that average rate of living cells using Method 2 （collagenas＋trypsin） was 94.4%, which had significant differences with those in Method 1 and 3. Survival time and rate of SSCs cloning in culture System Ⅳ were 45.5＋3.2 days and 31%＋16% respectively, which were extremely significant higher than those in another five culture systems. SSCs were subcultured to the 3rd passage, and rates of cloning of passage Ⅰ , Ⅱ and Ⅲ were 31.6%, 20% and 18% respectively. SSCs formed cell clonies and proliferated, and cell clones of SSCs were positive to alkaline phosphatase （AKP） and stage specific embryonic antigen- 1 （SSEA-1）. SSCs of the 3rd passage still maintained normal chromosome karyotype. Conclusion.. The SSCs were suitable to be isolated using combination of collagenase with trypin and subcultured in culture System/V.

关 键 词：鸡 精原干细胞 分离 体外培养 

分 类 号：Q813.1[生物学—生物工程]