摘要
目的:探讨人精原干细胞分离、纯化及以人胚胎成纤维细胞为饲养层培养的方法和条件。方法:利用两步酶法和Percoll不连续密度离心法分离、纯化人精原干细胞,在人胚胎成纤维细胞饲养层上培养;用免疫组织化学方法检测精原干细胞表面标志SSEA-1和OCT4;检测精原干细胞克隆碱性磷酸酶活性;逆转录聚合酶链反应(RT-PCR)检测精原干细胞相关基因的表达。结果:精原干细胞在人胚胎成纤维细胞饲养层上可以存活并增殖形成集落。集落未分化标志检测显示SSEA-l、OCT4呈阳性,碱性磷酸酶活性呈强阳性,并表达精原干细胞相关基因。结论:人胚胎成纤维细胞饲养层可以支持人精原干细胞的生长。
Objective： To investigate the methods and conditions for the isolation, purification and culture of human spermatogonial stem cells （SSCs） on the feeder layer cells of human embryonic fibroblasts （hEFs）. Methods： SSCs isolated and purified from normal human fetal testicular tissues by sequential two-step enzyme digestion and Percoll uncontinuous density gradient centrifugation were cultured on the feeder layer cells of hEFs isolated from 5-9 weeks old human embryos. The surface markers SSEA-1 and OCT4 of the SSCs were detected by immunohistochemistry; the alkaline phosphatase （AKP） activity of the SSC clones measured; and the expressions of the SSC-related genes determined by RT-PCR. Results： SSCs survived, proliferated and formed colonies on the feeder layers, and the colonies were highly positive for SSEA-1 and OCT4, with strong AKP activity and high expressions of the SSC-related genes. Conclusion ： The feeder layer of hEFs supports the growth of human spermatogonial stem cells.
出处
《中华男科学杂志》
CAS
CSCD
2008年第12期1063-1068,共6页
National Journal of Andrology
基金
上海市计划生育委员会科研基金(2007JG06)
关键词
精原干细胞
人胚胎成纤维细胞
饲养层
分离
纯化
细胞培养
人
spermatogonial stem cell
human embryonic fibroblast
feeder layer
isolation
purification
cell culture
human
