Establishment Of Sheep Immortalized Epididymal Epithelial Cell Line And Analysis Of Its Biological Characteristics

The epididymis is an important reproductive organ in male mammals.The expression of the epididymis specific genes and protein secretion contribute to the formation of the epididymis microenvironment.During sperm mature,transport,and store in the epididymis,a variety of cytokines secreted by epididymal epithelial cells play an important role in sperm ability of motility and fertilization.The regulation of epididymal function is generally studied by primary epididymal epithelial cells,but the limited proliferation of primary cells limits many long-term studies.Therefore,in this study,primary epididymal epithelial cells of sheep were obtained by enzymatic digestion,and the human telomerase reverse transcriptase(hTERT)was transferred into cells to establish an immortalized epididymal epithelial cell line,which will provide a basis for the further basic scientific research.1.The culture of sheep primary epididymal epithelial cells.The healthy testis of the sheep was collected,then the tunica albuginea was removed,and the primary cells were isolated and cultured after treated by the 0.1%collagenase IV.The results showed that the sheep primary epididymal epithelial cell line had high purity,clear boundary and close cells connection.The cells morphology looked like cobblestone.The epididymal epithelial cells can be subcultured for up to 6 passages,which the epithelial cell-specific marker keratin 18 can be steadily expressed,also the epididymal epithelial cells can be grow successfully after frozen-thawed.2.The pCI-neo-hTERT was transferred into the sheep epididymal epithelial cells(SEECs)by liposome and the hTERT-SEECs was obtained by G418 screened.The keratin 18(CK18)and hTERT were identified by immunofluorescence;The expression of hTERT mRNA was detected by RT-PCR;The growth curvewas detected by cck-8;The growth cycle and apoptosis status were detected by flow cytometry;The ploidy was detected by karyotype analysis;The mRNA and protein expression of glutathione peroxidase 5(GPX5)and androgen receptor(AR)were investigated.The results showed that the hTERT was transferred successfully into the SEECs;After 45 passage,hTERT-SEECs could express CK18 and hTERT as well as were in normal diploid;the proliferation ability of hTERT-SEECs was significantly higher than that of SEECs(P<0.05),hTERT-SEECs in S phasewere significantly higher compared to SEECs(P<0.05),and the living cells rate of hTERT-SEECs was significantly higher than that of SEECs(P<0.05),the late apoptosis rate of hTERT-SEECs was significantly lower than that of SEECs(P<0.05);hTERT-SEECs could steadily express GPX5and AR after long timeculture.In conclusion,A effective culture produre for sheep primary epididymal epithelial cells was establishedin this study,and the sheep immortalizedepididymal epithelial cell linewas established sucessfully.The established hTERT-SEECs have strong proliferative capacity and retain the biological characteristics of epididymal epithelial cells,which can provides basis for further researches.