Effect Of EP₂ Receptor Agonist Butaprost And Estrogen On Expression Of TGF-β3 And LIF In Bovine Oviduct Epithelial Cell

Objective:The study aims to reveal the estrogen, prostaglandins, prostaglandin receptor pathway of secretion of transforming growth factor beta 3 (TGF beta 3), leukemia inhibitory factor (LIF) on oviduct epithelial cells have any synergistic regulation. The experiment provide a theoretical basis for clinical rational drug use in treatment of oviduct infertility caused by dysfunction and improve the reproduction capability.Methods:1) Using the method of trypsin digestion and mechanical method successfully develop bovine oviduct epithelial cells and to experiment with the fourth generation of cell types.2) Real-time PCR were used to detect TGF-β3, LIFmRNA expression in bovine oviduct epithelial cells after (estrogen, butaprost, indomethacin) treatment at indicated time points (2h,4h,8h.16h,24h,48h).3) Use different concentrations of Butaprost (10-9-10-5mol/L) and estrogen (10-10mol/L) to treat the bovine oviductal epithelial cells, and then use the real-time PCR to detect the expression of TGF beta 3, LIFmRNA.4) Using Western Blot method to detect the secretion of TGF-(33 in bovine oviduct epithelial cell after (estrogen, butaprost, indomethacin) treatment.5) Using ELISA method to measure the expression of LIF level in bovine oviduct epithelial cells.Results:first of all, the successful cultivation of bovine oviduct epithelial cells in vitro. Secondly, After treatment with estradiol,the expression of TGF-β3 at 16,24 and 48 h compared with blank group were extremely significantly (P<0.01) increased, TGF-β3 mRNA expression reached minimal at 4 h respectively;expression of estrogen effects of 16,24 and 48 h of oviduct epithelial cells TGF beta 3 and blank group group were significantly increased (P< 0.01), decrease the expression of H 4 was significant (P< 0.05); and the effect of EP2 receptor agonist butaprost and estrogen coordinated regulation of TGF beta 3. Addition of indomethacin can inhibit the effective of endogenous prostaglandin for TGF beta3 expression. After treatment with estradiol (without indomethacin pretreatment) in oviduct epithelial cells,the expression of LIF at 2 h compared with blank group significantly increased (P< 0.01), and 2 h the expression of LIF mRNA reached the maximum. Indomethacin pretreatment,the expression of LIF mRNA compared with no indomethacin treated group was increased. Third, With different concentrations of butaprost to stimulate the oviduct epithelial cells, the TGF-β3 and LIFmRNA expressed in a concentration dependent manner.Fourth, Western Blot to detect the expression of TGF beta 3 and TGF beta 3 mrna test results are basically identical. Fifth, ELISA detection of LIF in bovine oviduct epithelial cells, the results are in conformity with LIFmRNA test results.Conclusion:(1) Estrogen can promote the expression of cytokine TGF-β3、LIFmRNA in bovine oviduct epithelial cell.(2) Estrogen and EP2 receptor agonist Butaprost have synergistic regulation of cytokine TGF-β3 in bovine oviductal epithelial cells, have antagonistic effect of cytokine LIF in bovine oviductal epithelial cells.(3)Endogenous prostaglandins promoted the expression levels of TGF-β3 and inhibited the expression levels of LIF in bovine oviductal epithelial cells.