Establishment Of Immortalized Bovine Endometrial Luminal Epithelial Cells

Dairy cattle breeding disorder seriously influence the development of scaled dairy farming. Pregnancy failure is the important factor causing the cow production performance. When embryo forms in the womb, it start to induce maternal signal, causing the mother′s immune system response. In a series of pregnancy immune toleration reaction, it ensure the normal implatation, growth and development of fetus. Bovine endometrial luminal epithelial cells in vitro play an important pole in study of cow breeding physiology. But because of slowly limited proliferation and strict environment from the separation and cultivation of primary cell in vitro, which seriously affected the development of basic scientific research.1 The experiment using healthy endometrial tissue to cultivate and purify the endometrial luminal epithelial cells of original generation, continuing to develop. We transfect the exogenous gene carrier of the hTERT into 3 or 4 generation cells in good condition, cell stable expression, screened positive cells continuing to develop. The immortalized cells is bovine endometrial luminal epithelial cells(imEECs), not only retain the biological activities of origination generation, also has the ability of relatively infinite proliferation, which laid a foundation for subsequent pregnancy immune mechanism research.2 The primary cells are cultivated by way of combination of tissue cultivation and pancreatic cultivation. The cells climb out of tissue about four days, they are round or oval irregularly, which are compactly connected each other, well-defined, positively stained by specificity keratin 18.3 The third generation of cells are transfected into the plasmid pCI-neo-h TERT by liposome. After screening by 500 ug/m L G418, positive cells are developed. The cells has spread to 70 generations in a good condition, keratin 18 dying is still positive. Using immunofluorescence method to detect the expression of hTERT in hTERT-BEECs at 30 and 50 generations, which showed positive. However, primitive cells were negative. It was indicated that exogenous gene was successfully transfected into cells and could express normally.4 Using IFN-τ to deal with EECs and hTERT-BEECs respectively, RT-PCR was used to detect the expression of MX1, MX2, IRF1, IRF2 and IRF9, which showed all positive. hTERT-BEECs retain the biological activity in contrast to primary cells, which could undertake following experiment.Conclusion: making the exogenous gene hTERT transfect into EECs, the bovine endometrial luminal epithelial cells we acquired are immortalized and retain biological activities, which are used to clarify the mechanism of pregnancy for cattle.