| Chapter?Changes of CD4+CD25+Foxp3+regulatory T cell in different stages of pregnacyObjective:To evaluate the proportional changes of CD4+CD25+Foxp3+regulatory T cell (Tregs) and Foxp3 mRNA expression in maternal peripheral blood during the first, second and third trimesters.Methods:This study was conducted on pregnant women at the Third Xiangya Hospital, Central South University, obstetric clinic regularly for check-ups between August 2009 and June 2010. A total of 60 pregnant women were enrolled, of which each 20 were within the first, second and third trimesters, and we choose 20 healthy non-pregnant women for controls. Fresh peripheral venous blood samples were obtained from all the 80 subjects. Peripheral blood CD4+CD25+Foxp3+Treg cells was analyzed by 3-color flow cytometry. Foxp3 mRNA expression in peripheral blood mononuclear cells was measured by semi-quantitative RT-PCR.Result: 1. In maternal peripheral blood the percentage of CD4+CD25+Foxp3+ Treg cells in CD4+T cells were 6.39 (6.04-8.33)%,7.90 (6.18-9.30) %,7.46 (6.59-7.94)% during the first, second and third trimesters. The controls was 3.08 (2.66-3.30)%. Notably, the percentage of circulating CD4+CD25+Foxp3+Treg cells was significantly increased in women during the first trimester compared with controls (P<0.01), peaking during the second trimester and then slightly declining in the third trimester. No significant differences in the proportion of Tregs cells were detected among the pregnant women at different gestational ages (P>0.05).2. Foxp3 mRNA expression in PBMC during the first, second and third trimesters was significantly higher than controls, respectively [(0.564±0.138), (0.649±0.126), (0.602±0.088) vs (0.307±0.052)] (all P<0.01). There was no significant statistical difference among the three groups at different trimesters (P> 0.05), and we found that the trend of Foxp3 mRNA expressions are consistent with the that of CD4+CD25+Foxp3+ Treg cells in PBMC, that is, Foxp3 mRNA expression and the proportion of CD4+CD25+Foxp3+Treg cells significantly increased after pregnancy, reached a peak at the second trimester, and a slightly lower at the third trimester.Conclusion: 1. Foxp3 mRNA expression and the proportion of CD4+CD25+Foxp3+ Treg cells changed in the same trend in maternal circulation, suggesting that Foxp3 is a powerful marker for deteciton of Treg cells.2. The significant elevation of maternal circulating CD4+CD25+Foxp3+ Treg cells after pregnancy suggests an important role of Treg cells in maintenance of pregnancy.Chapter?Changes of CD4+CD25+Foxp3+regulatory T cell during labor at term and pretermObjective:To evaluate the proportional changes of CD4+CD25+Foxp3+Tregs cells and Foxp3 mRNA expression in peripheral blood, decidua Foxp3 mRNA and protein expressions during labor at term and preterm.Methods:This study was conducted in pregnant women admitted in the Third Xiangya Hospital, Central South University and at obstetric clinic regularly for check-ups between August 2009 and June 2010. A total of 99 pregnant women were enrolled, of which 28 at term with labor,20 without labor and 51 were at 28-36 weeks of gestation (16 with threatening and 15 actual preterm labor and 20 with normal pregnancy as controls). Peripheral blood CD4+CD25+Foxp3+Treg cells was analyzed by 3-color flow cytometry. Foxp3 mRNA expression in PBMC and deciduas was measured by semi-quantitative RT-PCR. Foxp3 protein expression in deciduas was analyzed by Western Blot. The placenta and fetal membrane in patients eventually delivered preterm was examined by HE staining.Result:1. The proportion of CD4+CD25+Foxp3+Treg cells was significantly lower in women at term in labor relative to those at term without labor [3.52(2.98-4.16)% vs 7.46(6.59-7.94)%, P<0.01].2. Foxp3 mRNA expression in maternal PBMC was significantly lower in women at term in labor relative to those at term without labor [(0.312±0.050) vs (0.602±0.088), (P<0.01)].3. Foxp3 mRNA and protein expressions in deciduas were significantly lower in women at term in labor relative to those at term without labor [(0.249±0.085) vs (0.556±0.090), (0.280±0.078) vs (0.579±0.093), both P<0.01].4. Six of 16 cases of threatened preterm labor eventually delivered preterm, and patients admitted as preterm labor all delivered preterm. Placenta of 16 (76%) in the total of 21 patients delivered preterm were examined with histologic chorioamnionitis.5. In maternal peripheral blood the percentages of CD4+CD25+Foxp3+ Treg cells in CD4+T cells were 5.27 (3.82-7.44)%,1.63 (1.09-2.73) %,7.38 (6.90-8.78)% in threatened preterm labor group, preterm labor group and the controls, showing the proportion of Treg cells significantly decreased between the preterm labor group and threatened preterm labor group (P<0.01), or between the threatened preterm labor group and the controls(P<0.01). Preterm women with evidence of chorioamnionitis had a significantly lower proportion of CD4+CD25+Foxp3+Treg cells than those without chorioamnionitis in preterm deliveries [1.68 (1.15-2.68)% vs 3.34 (2.71-5.05)%, P<0.01]. Preterm women inrespective of the histological chorioamnionitis also had a significantly lower proportion of CD4+CD25+Foxp3+Treg cells than controls (P<0.01), but no significant difference were found when compared with that of the term labor group (P>0.05).6. Foxp3 mRNA expressions in maternal PBMC were significantly lower in patients admitted with preterm labor than those with threatened preterm labor [(0.168±0.085) vs (0.477±0.156), P<0.01], Foxp3 mRNA expression in maternal PBMC was also significantly lower in patients admitted with threatened preterm labor than controls (0.632±0.092), (P<0.01).7. Preterm women with evidence of chorioamnionitis had a significantly lower Foxp3 mRNA and protein expression in deciduas than those without chorioamnionitis in preterm deliveries [(0.146±0.020) vs (0.228±0.016)?(0.141±0.030) vs (0.246±0.030, both P<0.01], while when compared deciduas Foxp3 mRNA and protein expression in preterm women without evidence of chorioamnionitis with those of term labor group, it showed no significant difference (both P>0.05).Conclusion:The significantly decrease of CD4+CD25+Foxp3+Treg cells in peripheral blood of term and preterm women suggests weakened negative regulatory immune function may be associated with the oneset of labor.Chapter?Study on PD-1 neutralizing antibody to Mice's Age and Delivery Interval and Its MechanismObjective:To evaluate PD-1 mediated CD4+CD25+Treg cells in immune regulation of pregnant mice's age and initiaton of labor by using PD-1 neutralizing antibody.Methods:1. Establish patterns of pregnant mice the gestational age of which is precise and divide them into three groups randomly which are PD-1mAb group, IgG group, physiological saline(NS) group, injected(i.p.) with PD-1mAb 250?g, IgG 250?g, NS 250?g respectively on the 5th,8th,11th,14th days of pregnancy.2. Isolate splenic mononuclear cells from six non-pregnant mice and six pregnant mice; examine the proportion of CD4+CD25+Treg cells in CD4+T cells and PD-1 expression in Treg cells by flow cytometry.3. Observe the PD-1 neutralizing antibody to Mice's Age and Delivery Interval.4. Using semi-quantitative RT-PCR and Western blot to measure the influence of PD-1 neutralizing antibody on Thl cytokines (TNF-a, IFN-y) and Th2 cytokines (IL-4, IL-10) mRNA and protein expression levels in placenta of pregnant mice.Result:1. The proportion of CD4+CD25+Treg cells in splenic mononuclear cells significantly increased when compared the pregnant mice with non-pregnant ones [(13.89±1.69)% vs (5.88±0.84)%, P<0.01]. No significant statistic difference was found when compared the expression of PD-1 on CD4+CD25+Treg cells before and after pregnancy [(6.41±0.90)% vs (7.40±0.86)%, P>0.05].2.The influence of PD-1 neutralizing antibody on gestational age:The gestational age of mice in PD-1 mAb, IgG, NS groups were (19.74±0.40), (20.14±0.45), (20.00±0.26) days respectively. No preterm labor occurred. The gestational age of mice in PD-1mAb group was shorter than other groups, but with no significant difference (P>0.05).3.The influence of PD-1 neutralizing antibody on delivery interval of pregnant mice:The delivery interval of mice in PD-1mAb, IgG, NS groups were (5.78±1.68), (7.86±1.58), (8.03±1.64) minutes respectively. It was significantly shorter in PD-1 mAb group than IgG or NS groups (both P<0.05), while no significant statistic difference was found between IgG and NS groups (P>0.05).4.The possible mechanism about dilevery interval of pregnant mice affected by PD-1 neutralizing antibody:1) IL-4, IL-10, TNF-a and IFN-?mRNA relative expression in placenta of pregnant mice in PD-1mAb, IgG, NS groups were (0.13±0.03,0.17±0.04,0.18±0.04), (0.14±0.05,0.20±0.02,0.21±0.04), (0.39±0.06,0.29±0.06,0.31±0.07), (0.36±0.07,0.27±0.04,0.26±0.04) respectively. IL-4mRNA, IL-10 mRNA expressions in PD-1mAb was significantly lower than IgG or NS groups (both P<0.05). While TNF-a mRNA, IFN-y mRNA expressions in PD-1 mAb group were significantly increased than IgG or NS group (both P<0.05).2) IL-4, IL-10, TNF-a and IFN-y protein relative expression in placenta of pregnant mice in PD-1 mAb, IgG, NS groups were (0.13±0.02,0.22±0.04,0.19±0.04),(0.16±0.04,0.22±0.03,0.21±0.05), (0.41±0.06,0.33±0.05,0.31±0.07),(0.36±0.06,0.29±0.04,0.28±0.04) respectively. IL-4, IL-10 protein expressions in PD-1 mAb were significantly lower than IgG or NS groups (both P<0.05). While TNF-?, IFN-y protein relative expression in PD-1 mAb group were significantly increased than IgG or NS groups (both P<0.05).Conclusion:1. CD4+CD25+regulatory T cells can express PD-1.2. PD-1 neutralizing antibody may affect PD-1-mediated CD4+CD25+ Treg cells in immune regulation, which led to the imbalance of Thl/Th2 cytokines, thus accelerate the delivery process. |
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