Effect Of HCG On Regulatory T Cells And Its Role In Immune Tolerance In Pregnancy

BackgroundPregnancy is the whole process of the implantation of fertilized eggs to the end of fetus,and the early fertilized eggs are used as antigenic components to stimulate the maternal placenta and even the whole body.In the early stage,the maternal immune tolerance to ensure the normal development of the fetus,the establishment of the process involves a variety of immune cells and immune molecules response,the need for coordination and balance between the reactions.It is not clear that the mechanism of immune tolerance in pregnancy is not fully understood.Most of the researches at home and abroad have been concentrated on the role of placenta,such as local innate immunity and adaptive immunity.CD4+ T cells are an important cell body with the adaptive immune response.Recently,the researchers found a new group of T cells,which can produce negative cytokine interleukin-10 that play a role in immune regulation.IL-10 can regulate the balance between helper T 1(Th1),Th2 and Th17.Our previous study group members found the content of serum IL-10 of pregnant women was significantly increased,and decreased in late pregnancy period,combined with human chorionic gonadotropin pregnancy increased hCG,we further analyze the relationship between hCG and IL-10,analysis of regulatory T cells proportional change is affected by steroid hormones during pregnancy,and to verify the effect of hCG on the regulation of the differentiation of T cells in vitro.On the one hand,this study can help us to understand the mechanism of the formation of immune tolerance in pregnancy,on the other hand,it can provide the theoretical basis for the abortion,fetal dysplasia and other diseases.At the same time,we also study the relationship between IL-10 and immune tolerance,and provide an important immune target for inducing immune tolerance to maintain the survival of new organ.Objective:This study aimed to analyze the content changes of peripheral blood hCG and IL-10,and then analyze the relationship between them.In vitro,we verified that hCG could promote regulatory T cell differentiation,which may make clear the mechanism of pregnancy immune tolerance and provide a theory basis for screening effective drugs to many pregnancy related disease.Methods:1.Research object: according to the standard of "obstetrics and Gynecology",the pregnant women were selected in the Department of gynecology and obstetrics.The experimental group was included in the early,middle and late stage of pregnancy,and the control group was included in the healthy pregnant women.2.Sample collection and pretreatment with heparin sodium anticoagulation tube(green)from the experimental group and the control group of women 10 ml in peripheral blood,then centrifuged and frozen,mononuclear cells were isolated by density gradient centrifugation,for subsequent experiments.3.The content of hCG in plasma samples was detected by SIEMENS 2000 biochemical analyzer based on the chemiluminescence method.4.The level of supernatant IL-10 was detected: in the presence or absence of hCG,PMA was used to stimulate healthy human PBMC,and the content of IL-10 in culture supernatant was detected by IL-10 ELISA kit.5.Detection the level of IL-10 mRNA: in the presence or absence of hCG,PMA was used to stimulate healthy human PBMC,and then we extracted total RNA,reversed transcription,detected IL-10 mRNA expression by real-time fluorescence quantitative PCR.6.Detection the proportion of CD4+CD25+IL-10+Treg cell: in the presence or absence of hCG,PMA was used to stimulate healthy human PBMC,and the ratio of regulatory T cells in PBMC was detected and analyzed by multicolor fluorescent flow cytometry.Results:1.Chemical chemiluminescence detection results showed that hCG in the early,middle and late in pregnancy were(51304.3±27601.1)IU/L,(14310.2±17719.3)IU/L and(13257.4±7031.5)IU/L,which were all higher than that of non pregnant women peripheral blood hCG content(2.8±3.3)IU/L,and there was significant difference.2.The results of ELISA showed that IL-10 in the early and middle stages of pregnant women were(326.8±117.0)pg/ml and(213.8±99.1)pg/ml,significantly higher than those of non pregnant women(91.3±11.1)pg/ml,and there was statistical difference.The content of IL-10 in late pregnancy was(150.9±18.8)pg/ml,which was higher than non pregnant women(P>0.05).IL-10 was highly correlated with hCG(R2=0.719,P<0.001).3.The peripheral blood PBMC of healthy non pregnant women was stimulated by hCG,in presence of hCG,the IL-10 content in culture supernatant was(24.2±9.4)pg/ml,the expression of IL-10 mRNA was(2522.3±784.3);in absence of hCG,the IL-10 content in culture supernatant was(13.8±3.4)pg/ml,the expression of IL-10 mRNA was(139.8±311.0);the former was significantly higher than that of the latter and there was significant difference.4.In presence of hCG,the proportion of CD4+CD25+IL-10+ Treg was(7.8±2.1)%,in absence of hCG,the proportion was(4.5±1.8)%,the former was significantly higher than that of the latter and there was significant difference.Conclusions:1.Pregnancy peripheral blood plasma IL-10 content and hCG content are highly positive correlated,both increas in the early and reach the peak,and then begin to fall,which is similar to non-pregnancy women at the end of pregnancy.2.hCG can stimulate the proliferation of CD4+CD25+IL-10+ Treg cells,which secrete more IL-10 and then participate in the formation of maternal immune tolerance in pregnancy,and provide the necessary conditions for the normal growth and development of fetus.