鸡胚原始生殖细胞生物学特性鉴定及相关基因表达研究

Study on identification of chicken primordial germ cells and expression of specific genes

采用酶消化法分离鸡胚原始生殖细胞(PGCs),纯化后进行体外培养,传至5～6代时,通过对阶段特异性胚胎抗原-1(SSEA-1)免疫荧光标记、碱性磷酸酶检测等方法联合鉴定其基本生物学特性,同时以RT-PCR方法检测其特异基因的表达。结果表明:体外培养的PGCs维持未分化状态,碱性磷酸酶阳性、免疫荧光检测其特异标志物SSEA-1阳性。鸡胚PGCs表达减数分裂前标志基因Dazl、Nanog和GDF3,生殖细胞分化后期基因CVH,原始生殖细胞标志基因Blimp-1以及干细胞多能性相关基因Oct-4,不表达减数分裂启动基因Stra8。提示所获得的鸡胚PGCs,其生物学特性稳定,表达相关特异基因,为鸡胚PGCs的体外培养及鉴定提供理论依据。

Primordial germ cells（PGCs） were separated by EDTA-trypsin collection methods and cultured in vitro.The characteristics of the 5-6th passage of chicken PGCs was analyzed with alkaline phosphatase（APK） staining and immunofluorescent labeling of stage specific embryonic antigen-1（SSEA-1）,was a promising the expression of germ cell differentiation associated genes on PGCs was detected by RT-PCR.The results showed that positive labeling of AKP and SSEA-1 suggested PGCs maintained undifferentiated state.The specific marker genes of PGCs,Blimp-1,Oct-4,Dazl,Nanog,GDF3 and CVH were detected,but Stra8 was not detected in obtained PGCs.In vitro cultured PGCs have the characteristics of pluripotent stem cells,at the same time the specific marker genes of PGCs were expressed,thus we successfully obtained the chicken PGCs,which was a promising a source for in vitro culture and identification of chicken PGCs.