Expression Of IL-10 MRNA And Breg Cell Subsets In Different Gestational Period

ObjectiveRegulatory B cells, a new subset of B cells discovered in recent years, act as a negative regulator of the inflammatory and immune diseases mainly through the production of interleukin (IL)-10 and transforming growth factor beta (TGF-β). The establishment of immune tolerance in pregnancy is an extremely complex and coordinated process. The domestic and foreign scholars have done a thorough exploration of the mechanism of this immune tolerance, and most of them think that the body regulates the implantation mainly through local immune function. Based on the study that plasma IL-10 increased in the early pregnancy, we detect IL-10-producing B cell in different gestational period by using ELISA, real-time fluorescence quantitative PCR and flow cytometry. We explore the molecular mechanism of pregnancy tolerance which will provide a theoretical basis for the study of the pathogenesis of many diseases, such as abortion, intrauterine growth retardation, pregnancy induced hypertension syndrome and so on. Moreover, it can also provide a new way for the rejection of allograft rejection and the induction of immune tolerance.ObjectiveWe aim to detect the changes of regulatory B cells during different gestation period, and to investigate the regulation of immune tolerance in pregnancy.Methods1. Subjects investigated:in accordance with the 8th edition of "Obstetrics and Gynecology", we choose different pregnancy women in Zhu Ma-dian City People’s Hospital for the research object, according to the different period of pregnancy were divided into three groups:Early trimester, Second trimester and Third trimester. At the same time, the healthy no-pregnant women are chosen as a control.2. The collection and processing of samples:Peripheral blood mononuclear cells were collected from different pregnant women and healthy no-pregnant women and the separated plasma are stored in -80℃refrigerator for ELISA.3. Detection the level of plasma IL-10:using human IL-10 ELISA kit to detect the level of plasma IL-10.4. Detection the mRNA expression of IL-10 subunit:extract the PBMC total RNA of different pregnant women and healthy no-pregnant women. After reverse transcription, the expression of IL-10 mRNA were detected by real-time quantitative PCR.5. Detection regulatory B cells and their subsets:PBMC was isolated from women of the same period of pregnancy and no-pregnant women. The distribution of regulatory B cells and its subsets were detected and analyzed by using fluorescent antibody combined with flow cytometry.6.Measurement data with standard deviation of mean and subtract (x-s), compare the difference between the two groups using t test, the data were compared by chi-square test. Inspection level of alpha=0.05, P<0.05 for the difference was statistically.Results1. Specimen groups:early pregnancy women (<13 weeks) 20 cases, the middle of a pregnancy women (14 weeks-27 weekend) 20 cases, women in late pregnancy (>28 weeks) 20 cases, women in healthy non-pregnant 20 cases, a total of 80 cases of samples.2.The results for ELISA:The plasma IL-10 levels were (326.85±33.91) pg/ml and (213.76±66.10) pg/ml in early and second pregnant women, respectively. They were higher than those in non pregnant women, and there were significant differences in the IL-10 levels in the early and mid pregnancy by t-test.3. The results for real-time quantitative PCR:The relative expression of IL-10 in early pregnancy was the highest, up to (3712.00±123.00) and the expression of IL-10 decreased with the extension of the gestation period.4. The results for flow cytometry:The percentage of CD19+IL-10+ B cells in the early and mid pregnancy were (14.30±2.12)% and (7.53±1.11)%, significantly higher than those in the late pregnancy and non pregnant women, and there were significant differences by chi-square, then we found that the cells were highly expressed CD24 and CD38, but not CD1d and CD5.Conclusions1.The mRNA and protein expression of IL-10 were highly expressed during the gestation period, especially in the early and middle stages of pregnancy. Increased IL-10 was responsible for the negative regulation of and the establishment of immune tolerance during the gestation period.2. In another word, the CD19+CD24+CD38+IL-10+B cells may act regulation of immune tolerance in pregnancy.