| Objective:1.To investigate the expression of PD-1 and Tim-3 on monocytes in peripheral blood and the secretion of cytokines,such as IL-4?IL-6?IL-10?IL-12 and IFN-?,and to provide theoretical basis for research the pathogenesis of URSA.2.To investigate the regulation of Tim-3 and PD-1 on the function of monocytes,and to explore the possible mechanism of Tim-3 and PD-1 in URSA.3.Tim-3 or PD-1 may be a new target for the immunotherapy of URSA.Methods:Selected 30 healthy pregnant women as normal pregnancy group(NP group),30 cases early pregnant women who had a history of unexplained recurrent spontaneous abortion as URSA group.Separated peripheral blood mononuclear cells by density gradient centrifugation from peripheral vein blood,Flow cytometry was used to detect the frequency of Tim-3 and PD-1 on CD14~+monocytes of both groups.ELISA was used to detect the concentrations of IL-4?IL-6?IL-10?IL-12 and IFN-? in the plasma of both groups.Selected 10 cases of patients in both groups,sorted CD14~+monocytes by positive immune magnetic bead separation method and cultured with anti-Tim-3,anti-PD-1 or anti-Tim-3+anti-PD-1 overnight then stimulated by LPS/R848(5ug/mL)in vitro.Then FCM was used to detected the proportion of PD-1 or Tim-3 on CD14~+monocytes and the ratio of IL-12 in CD14~+monocytes.Using GraphPad Prism5.0 statistical software to analyze datas and drawings.Results:1.Compared with normal early pregnancy group,the frequency of Tim-3 and PD-1expression on CD14~+monocytes in peripheral blood of URSA patients was decreased(P<0.05).Correlation analysis showed Tim-3 and PD-1 were positively correlated(r=0.6665,r=0.6324,P<0.01).2.The concentration of IL-4 and IL-10 in plasma of group URSA were decreased than NP group(P<0.01),while the concentration of IFN-?,IL-6 and IL-12 were up-regulated in URSA group(P<0.01).The correlation analysis suggested that IL-12 with IL-4 and IL-10 were negatively correlated(r=-0.6714,r=-0.5593,P<0.01);while IL-12 with IL-6 and IFN-? were positively correlated(r=0.7740,r=0.5590,P<0.01).3.The correlation analysis of Tim-3 and PD-1 with those cytokines showed that it is positively correlated with IL-4 and IL-10(r=0.6343/0.426,r=0.5356/0.4529,P<0.05),negatively correlated with IL-6 and IFN-?(r=-0.5153/-0.5649,P<0.05,r=-0.4518/-0.6543,P<0.01)and negatively correlated with IL-12(r =-0.5216,r=-0.540,P<0.01).4.The proportion of PD-1 expression on CD14~+monocytes was decreased after blocked Tim-3 in both groups,the ratio of PD-1 expression on monocytes is lower in URSA than that in NP group.Similarly,the proportion of Tim-3 expression on CD14~+monocytes was decreased after blocked PD-1(P<0.001).5.Compared with normal early pregnancy group,the frequency of IL-12 expression in CD14~+monocytes in peripheral blood of URSA patients was increased.The concentration of IL-12 expression in CD14~+monocytes was increased after blocked Tim-3 or PD-1 pathway.The expression of IL-12 in monocytes after blocked Tim-3 was higher than that in blocked PD-1 in normal early pregnancy group.But the URSA group had not this result.Compared with blocked Tim-3 or PD-1 pathway,the level of IL-12 in CD14~+monocytes was higher after blocked both pathways,the difference was statistically significant(P<0.01).Conclusion:1.Tim-3 and PD-1 are involved in the maintenance of normal pregnancy,the dysregulation of Tim-3 and PD-1 on monocytes may be related to the occurrence ofURSA.2.The balance of Th1/Th2 to Th1 in URSA.Tim-3 and PD-1 may indirectly regulate the secretion of cytokines by inhibited the secretion of IL-12 by monocytes,leaded to the equilibrium of Th1/Th2 to Th1,which was involved in the occurrence of URSA.3.By blocked Tim-3 and PD-1 signals in vitro,it was found that Tim-3 and PD-1inhibited the secretion of IL-12 by monocytes,which negatively regulated the function of monocytes and indirectly regulated Th1/Th2 balance. |
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