不同培养方法对鸡精原干细胞体外培养效果的影响  

Effect of Culture in vitro of Spermatogonial Stem Cells by Different Culture Systems in Chicken

作　　者：吴信生[1] 孙思宇[1] 吴洪[1] 魏彩霞[1] 孙国波[1] 余飞[1] 赵文明[1] 徐琪[1] 陈国宏[1] 李碧春[1] 

机构地区：[1]扬州大学动物科学与技术学院,扬州225009

出　　处：《畜牧兽医学报》2008年第9期1196-1203,共8页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金资助(30671509);国家高新技术"863"项目(2007AA100504)

摘　　要：本研究从孵化至16 d的鸡胚睾丸中分离获取精原干细胞(Spermatogonial stem cells,SSCs),比较3种培养液、2种处理(有无饲养层)对SSCs生长的影响,并对SSCs保持特性进行鉴定。结果表明:在无饲养层细胞存在的条件下,DMEM、TCM199和RPMI1640培养液中,SSCs的存活时间分别为6.5、6.0和3.5 d,DMEM和TCM199培养液之间差异不显著(P>0.05),但两者与RPMI1640之间均存在极显著差异(P<0.01)。在有饲养层细胞存在的条件下,在DMEM、TCM199和RPMI1640培养液中,SSCs的存活时间分别为45.5、38.0和14.0 d,三者相互之间存在着极显著的差异(P<0.01)。在6种培养体系中,SSCs在培养体系Ⅳ中的存活时间和AKP阳性克隆率分别为(45.5±3.20)d和0.31±0.46,极显著地高于其他5种培养体系(P<0.01)。SSCs在培养体系Ⅳ中传代至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AKP活性检测和SSEA-1免疫染色,均呈阳性。传代至第3代的SSCs,其染色体组型保持不变,为2n=78。这些结果表明,SSCs在以DMEM为基础培养基的培养体系Ⅳ中培养至第3代时,仍保持干细胞未分化特性。Spermatogonial stem cells （SSCs） were isolated from chicken embryo testis on 16th hatching days. Growth of SSCs which were cultured in vitro in three culture solutions with or without feeder layer were compared, and the characteristic of SSCs was identified. The results showed that, without feeder layer of fibroblast cells, survival times of SSCs in DMEM, TCM199 and RPMI1640 were 6.5, 6.0 and 3.5 d, respectively, and there were extremely significant differences among in DMEM, TCM199 and in RPMI1640 （P〈0.01）. With feeder layer of fibroblast cells, survival times of SSCs in DMEM, TCM199 and RPMI1640 were 45.5, 38.0 and 14.0 d, respectively, and there were extremely significant differences among in three culture solutions （P〈0.01）. Survival time and rate of SSCs cloning in culture system IV were （45.5±3.2）d and 0. 31±0.46, respectively, and those in culture system IV were extremely significant higher than those in other five cultures（P〈0.01）. SSCs were subcultured in the culture system IV to 3ra pas- sage, and rates of cloning of systems at passage 1, 2 and 3 were 31.6%, 20% and 18%, respectively. SSCs formed cell clonies and proliferated, and cell clonies of SSCs were positive to alkaline phosphatase （AKP） and stage specific embryonic antigen-1 （SSEA-1）. The 3nd passage SSCs that had been cultured in vitro not only maintained the undifferentiated state and normal diploid kary-otype （2n= 78）, but also kept the necessary characteristics of SSCs, which was subcultured in culture system Ⅳ with DMEM and feeder layer of fibroblast cells.

关 键 词：鸡 精原干细胞(SSCs) 体外培养 传代 

分 类 号：S831[农业科学—畜牧学] Q813.11[农业科学—畜牧兽医]