摘要
该研究优化了山羊精原干细胞（goat spermatogonial stem cells,g SSCs）培养体系,使山羊精原干细胞能在体外长期培养,维持自我更新的能力并保持未分化状态。取3~5月龄山羊睾丸,采用两步酶消法结合差速贴壁方法得到山羊精原干细胞悬液,分别通过形态学观察、碱性磷酸酶（alkaline phosphatase,AKP）染色、特异基因表达及蛋白质水平的分析对培养的细胞进行鉴定;并以山羊睾丸支持细胞（goat sertoli cells,g SCs）、小鼠胚胎成纤维细胞（mouse embryonic fibroblasts,MEFs）和层黏连蛋白（laminin,L）为饲养层,观察饲养层对山羊精原干细胞体外增殖的影响。结果表明,山羊精原干细胞体外增殖形成克隆簇,AKP染色呈阳性。经RT-PCR检测,Oct-4、C-myc、Cyclin D1、Ngn3和TERT等干细胞特异基因均有表达。细胞免疫组化结果显示,Oct-4、SSEA-1、α6-integrin、Vasa和Thy-1蛋白质呈阳性。克隆簇统计显示,在山羊睾丸支持细胞上形成的山羊精原干细胞（goat spermatogonial stem cells,g SSCs）克隆数与其他两组比较差异显著（P〈0.05）。山羊睾丸支持细胞饲养层上的精原干细胞可在体外传3~4代,培养时间为2个月。结果证明,通过两步酶消法和差速贴壁法可以分离获得山羊精原干细胞,且山羊睾丸支持细胞能够促进g SSCs的增殖。
This study developed an optimized stem cell culture method. With this method goat spermatogonial stem cells （gSSCs） can be efficiently isolated and cultured in vitro for a long time to maintain self-renewal and remain undifferentiated state. Single gSSC suspension was obtained from testis of 3-5 month old goat by two- step enzymatic digestion and differential adhesion. The gSSCs were identified by morphology observation, alkaline phosphatase （AKP） staining and cell immunohistochemistry. The gSSCs were co-cultured with the feeder layer of goat sertoli cells （gSCs）, mouse embryonic fibroblast （MEFs） and laminin. The results showed that gSSC colonies were formed in vitro, and were positive AKP staining. The gene expressions of Oct-4, C-myc, CyclinD1, Ngn3 and TERT were identified by RT-PCR in gSSCs. The protein levels of Oct-4, SSEA-1, α6-integrin were also identified by immunohistochemistry staining. According to statistics of gSSCs colonies, the results showed that the number of colonies on sertoli cell feeder layer was more than on the other two feeder layer, and the difference was significant （P〈0.05）. The gSSCs were cultured for 3-4 generations as well as 2 months in vitro on sertoli cell feeder layer. All the above showed that the gSSCs can be obtained by the methods of two-step enzyme digestion and differential attachment, and the cells can be well cultured in vitro for proliferation on the feeder layer of sertoli cell.
出处
《中国细胞生物学学报》
CAS
CSCD
2016年第7期836-842,共7页
Chinese Journal of Cell Biology
基金
国家转基因新品种培育重大专项(批准号:2011zx08008-003)
山东省现代农业产业技术体系羊产业创新团队项目(批准号:621231)资助的课题~~
