Spermatogonia! stem cells（SSCs） form the foundation for spermatogenesis and sustain male fertility.To explore the regulatory mechanisms of chicken SSCs generation,we obtained highly purified chicken embryonic stem cells（ESCs）,primordial germ cells（PGCs） and SSCs by fluorescence-activated cell sorting（FACS）.High-throughput analysis methods（RNA-Seq） were used to sequence the transcriptome level of these cells.Gene ontology and Kyoto encyclopedia of genes and genomes（KEGG） pathway enrichment were used to analyze RNA-Seq results.BMP4 was used to induce chicken ESCs differentiation to SSCs-like cells in vitro.The quantitative real-time（qRT）-PCR was used to detect the expression changes of the key genes.The results showed that 22 relevant critical pathways were found by RNA-Seq,one of them was the Janus kinase/signal transducer and activator of transcription（JAK/STAT） signaling pathway.Total of 103 related genes were detected in this pathway.Protein-protein interactions analysis found that 87 proteins were significantly related to 19 key proteins in this pathway.These 87 proteins were enriched in 21 biological processes and 18 signaling pathways.Moreover,during the differentiation of chicken ESCs to SSCs-like cells induced by BMP4 in vitro,JAK2 and STAT3 were activated.The qRT-PCR results showed that the expression trends of JAK2 and STAT3 were basically the same as in vivo.We concluded that JAK/STAT signaling pathway plays an important role in the process of chicken SSCs generation both in vivo and in vitro;it may achieve its function through multiple biological processes and other related pathways.
鸡胚胎干细胞(cES)以其全能性,在研究胚胎发育生物学和家禽育种等方面有巨大的应用前景。本实验旨在建立cES无饲养层培养体系和对LipofactiminTMLTX(脂质体)介导转染体系进行优化。采用药匙法从新鲜受精蛋中分离获取cES,接种于层粘连蛋白包被的培养皿,待形成大克隆后,用口吸管剥离法吸取cES克隆进行消化传代,并采用生化和免疫学方法鉴定其干细胞活性。设计质粒量800、900、1 000 ng 3个水平,质粒、脂质体比为1∶1.5、1∶2、1∶2.5体系转染cES。结果表明:无饲养层培养体系体外培养cES细胞能稳定传代至第6代,鉴定结果显示碱性磷酸酶阳性和阶段特异性胚胎抗原1阳性,指明克隆能保持未分化状态;质粒900 ng、质脂比为1∶2时,可获得最佳转染效率72%,为cES的脂质体介导外源基因的转染提供参考。
