The Experimental Animal Study Of TSA Induced CD4+CD25+Regulatory T Cells In The Treating Of Recurrent Spontaneous Abortion

Objective:During normal pregnancy,fetus can be viewed as allogeneic transplant which has not been rejected by its mother,which reflects the maternal-fetal immune tolerance.After pregnancy,the changes of immunocyte’s functions in maternal-fetal interface are critical for keeping balance of periphery and maternal-fetal immune tolerance.In this research, based on analyzing the relationship between treg and the occurrence of pregnancy loss, to investigate the mechanism and the effect of the adoptivetransfer of CD4+CD25+regulatory T cells(CD4+CD25+Treg)from the TSA induced mice on pregnant outcome of the abortion-prone mice. Furthermore,it is aimed to be helpful for analyzing the immunological mechanisms of RSA,and to provide the experimental basis and theoretical evidence for standardized adoptive immunotherapy.Methods:The experiments were performed in following two groups:using female CBA/J×male BALB/c matings as the normal pregnancy model and female CBA/J×male DBA/2J matings as the abortion-prone model. CD4+CD25+Treg were isolated by MACS(magnetic cell sorting), FCM(flow cytometry)is to analyse the purity of CD4+CD25+Treg. In order to achieve the stable and lasting expression of Foxp3, TSA is applied to the apparent heredity beautification of the special location of Gene Foxp3in CD4+/CD25-T for miscarried mice. Purified CD4+CD25+Treg cells were obtained from spleens of the virgin CBA/J mice and pregnant CBA/J mice at the14th day of gestation respectively, and then injected intravenously into the pregnant CBA/J mice mated with DBA/2J males at the4th day of gestation(time of implantation), then the embryo resorption rate was counted at the14th day of gestation.Results:1) After the interaction between TSA and CD4+CD25+Treg, There was a significant improvement in the proportion of CD4+CD25+Treg, and as the concentration of TSA increased progressively, the ratio of CD4+CD25+Treg gradually increased, the concentration of TSA was5μmol/L, the ratio of CD4+CD25+Treg was55.14%±0.31%, the induced rate displayed the highest. Comparison between groups, there was no significant difference between the study groups and the control group, except for the group whose concentration was0.1μmol/L, the difference of concentrations in the other groups compared with the control group were significant.(P<0.05or P<0.01)2)The embryo resorption rate of CBA/J pregnant rats was12.00%±3.17%in the normal pregnancy model, however, in abortion model, the embryo resorption rate was29.88%±5.38%, with statistically significant difference (P<0.05). In the natural abortion model, the embryo resorption rate of CBA/J pregnant rats without injection of CD4+CD25+Treg cell induced by TSA from tail vein was28.38%±3.63%, while the embryo resorption rate of CBA/J pregnant rats after injection of CD4+CD25+Treg cell induced by TSA from tail vein was reduced to12.63%±3.50%, which showed significant difference between them (P<0.05), but there was no significant difference compared with normal pregnancy model (P>0.05)Conclusion:1) Immune tolerance of Treg lymphocytes played a key role during normal pregnancy,and absence or lack of immune tolerance is an important mechanism of pregnancy loss.2)The adoptive transfer of TSA-induced CD4+CD25+regulatory T cells therapy can induce the maternal-fetal immuno-tolerance, improved significantly the embryo resorption rate of abortion-prone model and maintaining pregnancy.