食蟹猴精原干细胞体外培养体系的初步建立  

The Preliminary Establishment of in Vitro Culture System for Spermatogonial Stem Cells of Cynomolgus Monkey

作　　者：张秀娟[1] 李学家[1] 张守全[2] 饶军华[1] 张艳春[1] 季芳[1] 彭白露[1] 刘晓明[1] 

机构地区：[1]广东省昆虫研究所华南灵长类研究开发中心,广州510260 [2]华南农业大学动物科学学院,广州510642

出　　处：《动物学杂志》2011年第6期46-52,共7页Chinese Journal of Zoology

基　　金：广东省自然科学基金项目(No.10451026001004520);广州市重大科技专项计划项目(No.2010U1-E00811)

摘　　要：为了掌握食蟹猴(Macaca fascicularis)精原干细胞(spermatogonial stem cells,SSCs)体外培养生长特性,并建立其培养体系。手术法获得幼年期食蟹猴单侧睾丸,改良的两步酶消化法获得其细胞悬液,添加特定培养液进行体外培养,以碱性磷酸酶(AKP)染色鉴定培养细胞,并评价不同饲养层细胞和不同接种密度对SSCs体外培养的增殖效果。结果表明,含10%FCS等补充物的DMEM液可支持食蟹猴SSCs的体外存活与增殖,培养3～5 d可观察到SSCs集落,AKP染色呈阳性;以支持细胞为饲养层体外培养SSCs 3 d和7 d,SSCs集落形成率均显著高于皮肤成纤维细胞饲养层(P<0.05),且以1×105个/mlSSCs接种密度是较好的原代培养开始。该培养体系的初步建立可为体外研究非人灵长类精子发生机制和SSCs基因修饰提供便利。This study was designed to understand the growth characteristics of spermatogonial stem cells（SSCs） of cynomolgus monkeys（Macaca fascicularis）and establish an in vitro culture system.Unilateral testis from male cynomolgus monkey of juvenile stage was obtained by surgical operation,and testicular cells were collected by two-step enzymatic digestion method.Purified SSCs were cultured in a special medium in vitro and cultured SSCs were identified by alkaline phosphatase（AKP） dye.Meanwhile,clone formation rate was used to evaluate the effect of different feeder cells and different seeding densities on SSCs proliferation.The results showed that the culture medium consisted of DMEM supplemented with 10% FBS supported SSCs survival and proliferation in vitro.The typical clone clumps formed during 3-5 days and were positive for AKP dye.Judged from the observations 3 days and 7 days after culture,SSCs plated on Sertoli cells（SC） feeder layer performed better than on cynomolgus monkey skin fibroblast（CSF） cells feeder layer,with significant difference in clone formation rate（P0.05）,and 1×105 cells/ml SSCs seeding density was proper for primary culture.This culture system provides convenience for the research of spermatogenesis mechanism and germ-line cells modification by gene targeting technology in nonhuman primates.

关 键 词：食蟹猴 精原干细胞 体外培养 集落形成率 

分 类 号：Q813[生物学—生物工程]