| During the bovine pregnancy, the embryo could escape the rejection from maternal immune system as an "Allograft" was realized by the appearence of Major Histocompatibility Complex (MHC), which existed on the interface of maternal and fetus.There were2types of BoLA-A molecules, which were classical and non-classical. BoLA-A was known as classical BoLA-I molecule, which could deliver antigen to T lymphocytes and Macrophage. MIC1was known as non-classical BoLA-I molecule, which could activate the activity of NK molecules. It had been proved that pregnancy recognition signal IFN-τ of ruminant was secreted by trophoblast cells, and it was capable of regulating and controlling the expression of BoLA-A and MIC1, which could contribute to the recognition of embryo and construction of pregnancy. By applying estradiol and progesterone to trophoblast cells, the different phase expression characters of these two hormones and signal IFN-τ could be identified. Thus, the study of estradiol and progesterone effects on BoLA-A and MIC1molecule expression was potential to reveal the molecular mechanism of the regulation role of BoLA-I and control of pregnancy immunity etc.Fristly, the concentration gradient of estradiol were12.5pg/mL,25pg/mL and50pg/mL, and progesterone were2.5ng/mL,5ng/mL and10ng/mL, and3h,6h,12h,24h were selected. Used various concentrations of estradiol and progesterone acting on cow endometrial epithelial cells independently or jointly, and selected RNA was extracted from the cells within different settled time. In the same situation MIC1expression increased obviously (25pg/mL estradiol and2.5ng/mL progesterone), accompanying with100ng/mL IFN-τ acting on cow endometrial epithelial cells jointly, and the time points were settled at6h,12h and24h. RNA of the cells was extracted and real-time PCR method was used to measure gene expression of MIC1. Furthermore, two groups which had the most significant regulation were selected to ally for western bolt to dectect the secretion of MIC1and BoLA-A.(1) The jointly application of estradiol and progesterone could obviously increase the gene expression of MIC1after cultuling6h, but the effect of separate is not obvious and the affection decrease after6h. The results suggested that the effect of estradiol and progesterone respective on expression of MIC1in endometrial epithelial cells was no significant; however, the effect on endometrial epithelial cells in6h could significantly increase the expression of MIC1, but the expression of MIC1was not obviously dectcted in other groups. The result also showed that estradiol and progesterone could obviously upregulated the gene expression of BoLA-A after cultuling12h,12.5pg/mL estradiol,12.5pg/mL estradiol and5ng/mL of progesterone,50pg/mL estradiol and5ng/mL progesterone groups could significantly up-reglate the expression of BoLA-A (P<0.01). But the expression decreased significantly in25pg/mL estradiol and10ng/mL progesterone groups after culturing24h (P<0.01). The results suggested that the effect of estradiol and progesterone respective or combined on expression of BoLA-A was significant after cultured12h. But the expression of BoLA-A went down-reglating after cultured24h.(2) The result showed the jointly application of estradiol, progesterone and IFN-τ could increase the MIC1expression at6h. The results showed that the IFN-τ could play a coordinate role in regulating the MIC1expression with progesterone and estradiol on endometrial epithelial cells after culturing6h, it was not obvious in other time. It was showed that the expression of BoLA-A was up-regulated by using100ng/mL IFN-τ,12.5pg/mL estradiol and5ng/mL progesterone jointly After cultuling6h,12h and24h. The results showed after24h, the joint action of estradiol and IFN-τthan single role raised significantly (P<0.01), and the jointly effects of progesterone and IFN-τ than either of them respectively (P<0.01). On the other hand, the jointly usage of P4and IFN-τ could promoting the BoLA-A expression than P4alone (P<0.01). When E2, P4and IFN-τ were used together for endometrial epithelial cells, it was not that obvious about their effects until24h.(3) The results of above process show the consistence of MIC1and BoLA-A expression in both gene and protein levels.Conclusion:The jointly application of estradiol and progesterone could obviously increase the gene expression of MIC1after cultuling6h, but the effect of separate is not obvious. The effect of estradiol and progesterone respective or combined on expression of BoLA-A was significant after cultured12h. But the expression of BoLA-A went down-reglating after cultured24h. At6h, progesterone, estradiol and IFN-τ expressed MIC1synergy effect significantly, and24h collaborative express BoLA-A significant. MIC1and BoLA-A at the genetic level and protein expression level of the results are consistent. |
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