Establishment And Identification Of Immortem Sheep Endometrial Epithelial Cell Lines

Successful implantation of the embryo is essential to ensure pregnancy.Embryo attachment is complete when a free mature blastocyst attaches to the endometrium,invades the mesenchyme and forms the placenta.In order for this process to be completed successfully,the endometrium must be in a receptive state.Some of the cytokines present in the endometrium regulate endometrial cell differentiation and blastocyst implantation in an autocrine or paracrine manner.Therefore,in vitro study of endometrial cells is important to reveal the mechanism of embryo attachment process.The limited number of passages of primary cells cultured in vitro and the loss of cell viability due to aging and physiological function variation are not conducive to long-term studies.Objective: To establish an immortalized endometrial cell line to lay the foundation for studying the biological functions associated with endometrial cells during sheep embryo attachment.Method:(1)In this study,sheep endometrial epithelial cells(EECs)were isolated and purified by tissue block culture method combined with trypsin differential time digestion.Immunofluoresce nce method was used to identify the specifically expressed protein keratin 18(CK18).(2)The p CI-neo-h TERT plasmid was introduced into primary epithelial cells by liposome transfection,and the transfected cells were identified by immunohistochemistry and immunofluorescence,growth curve was determined by CCK8 and apoptosis was detected by flow cytometry,and h TERT protein expression was detected by Western blot.(3)IFN-τ treated sheep endometrial epithelial cell lines for 24 h.Progesterone prolongation related genes ISG15,RSAD2,CTSL,CXCL10,prostaglandin metabolism related genes PLA2G4 A,SLCO2A1,PTGS2 and lipid metabolism related genes FABP4,SLC27A1 and PPARG were expressed.Result:(1)Cells started to crawl out of the tissue block on the 3rd day with the tissue block culture method,and the cells were spread out in a single layer on the 5th to 6th day,and the cell morphology was observed under an inverted microscope as shuttle-shaped and pavement-like,with both epithelial and stromal cells growing intertwined.When cells were digested using 0.25% trypsin,the stromal cells rounded about 1 min 30 s with abscission,while the epithelial cells took 6 min to round with abscission.Using differential time digestion,purified sheep endometrial epithelial cells were successfully obtained.The results of immunofluorescence identification showed that the epithelial cells were positive for keratin and negative for wave protein,with a positive rate of >95%;the stromal cells were positive for wave protein and negative for keratin,with a positive rate of >95%.(2)The p CI-neo-h TERT plasmid was transfected into primary EECs,and the transfected cells were screened with G418 at a concentration of 500 μg/m L,and the concentration was reduced to 250 μg/m L after 14 d.The positive clone was obtained by continuing the screening for 3 d.The cells were expanded and named h TERT-EECs.Immunofluorescence results showed that h TERT-EECs expressed CK18.Immunohistochemical results showed that 20 th generation h TERT-EECs were similar to 5th generation primary EECs in cell morphology,while 10 th generation primary EECs had an increased cell gap and tended to have cell growth arrest.CCK8 assay growth curves and flow cytometry apoptosis assay showed that h TERT-EECs growth was similar to that of primary EECs.western blot The results showed that h TERT protein was expressed in h TERT-EECs and was higher than that of the primary cells.(3)It was found that IFN-τ treatment at 10 ng/m L significantly increased the expression difference of ISG15 transcript levels most significantly,compared with the 0 ng/m L treatment group,the expression of progesterone prolongation-related genes RSAD2,CTSL and CXCL10 was significantly increased after IFN-τ treatment at 10 ng/m L,and the expression of prostaglandin metabolism-related genes SLCO2A1 and The expression of prostaglandin metabolism-related genes SLCO2A1 and PTGS2 increased significantly,while the expression of PLA2G4 A was significantly decreased,the expression of lipid metabolism and other related genes FABP4 did not change significantly,while the expression of SLC27A1 and PPARG increased significantly.Conclusion: In this study,we obtained an immortalized sheep endometrial epithelial cell line that retained the biological properties of normal primary cells by introducing the exogenous gene h TERT,and used this as a basis to study cell adhesion and endometrial remodeling during IFN-τ attachment in sheep embryos.