PDGFRA基因对牛未成熟支持细胞体外增殖的影响

PDGFRA Gene Promotes the Proliferation of Immature Sertoli Cells in Vitro

为了解血小板衍生生长因子受体α(PDGFRA)对牛睾丸支持细胞(Sertoli cells,SCs)体外增殖的影响,以新生牛的睾丸组织(1 d)为研究材料,利用两步酶消化法和差速培养法分离和纯化睾丸SCs,选择标志基因检测的方法对SCs进行鉴定。利用PCR法检测PDGFRA基因在SCs中的表达,构建shRNA干扰载体转染SCs,利用实时荧光定量PCR和Western blot方法检测该基因的表达量,采用MTS法检测细胞增殖。结果表明:成功分离纯化了SCs,检测到PDGFRA基因在SCs中表达,成功构建了PDGFRA基因的shRNA载体。随PDGFRA基因表达水平降低,细胞增殖减慢。该结果可为更好地了解PDGFRA在雄性动物精子发生和生殖器官发育过程中发挥作用的机制奠定基础。

PDGFRA,as a transmembrane receptor,could be combined with PDGFs. It was involved in embryonic development,tissue formation and regulation of cell proliferation,and survival and chemotaxis. However,the role of PDGFRA on the development of bovine sertoli cells has not been reported. In this study,Sertoli cells were isolated from bovine newborn testes of 1 day. SCs were obtained and purified by two step enzyme digestion method and differential speed method,respectively.The interference plasmid shRNA was constructed,the mRNA and protein levels of PDGFRA gene（real-time PCR and western blot） were assessed after transfection. Meanwhile,cell proliferation was detected using the MTS assay. SCs was successfully separated and purified,and PDGFRA gene was found to have been expressed on SCs. Cell proliferation was inhibited after transfection of PDGFRA shRNA vector. The results will lay a foundation for a better understanding of the spermatogenesis and reproductive development mechanism in male animals.