The Role And Mechanism Of Progesterone On CD4~+CD25~+Foxp3~+Regulatory T Cells During Pregnancy

Chapter IRelationship between progesterone level and CD4+CD25+Foxp3+regulatory T cells in different stages of mice pregnancyObjective:By evaluating the level of blood progesterone concentration, proportional changes of CD4+CD25+Foxp3+regulatory T cells (Treg) from spleen and deciduas and its Foxp3mRNA and protein expression during different stages of mice pregnancy, the relationship between progesterone level and CD4+CD25+Foxp3+regulatory T cells will be explored.Methods:Normal pregnancy model of Kunming mice was built, male and female2:1was kept in the same cage, female’s vaginal suppository positive was defined as pregnant day0.6mice of the same gestational age was confined as a group, a total of six groups as:pregnant day0,4,8,12,18and one hour after delivery. Six healthy nonpregnant females mice were control group. For each group peripheral blood was taken for detection of progesterone concentration by ELISA (enzyme-linkedimmuno sorbent assay, ELISA). Flow cytometry were used to detect the proportion of CD4+CD25+Foxp3+Treg in CD4+T cells from spleen and decidua. RT-PCR was used to detect Foxp3mRNA expression, and Western blot was used for detection of Foxp3protein expression levels.Results:1. In mice peripheral blood the concentration of progesterone were5.89±1.45、20.21±0.74、32.00±1.15、37.84±2.57、38.22±1.27and6.26±1.09ng/ml in pregnant Day0、4、8、12、18and postpartum, nonpregnant group was3.69±0.02ng/ml.Notably, the level of progesterone was significantly increased from D4with controls (P <0.05), peaking during the D12and D18and immediately drops after labor (P<0.05)2. In mice peripheral blood the proportion of CD4+CD25+Foxp3+Treg and its Foxp3mRNA expression level significantly increase from D4with controls (P<0.05), peaking during the D12and D18. Despite Treg proportion slightly declines, its Foxp3mRNA level decreases immediately after labor (P<0.05) which in consistent with the change of blood progesterone changes.3. In mice deciduas the proportion of CD4+CD25+Foxp3+Treg and its Foxp3mRNA expression level increase from DO with controls (P <0.05), peaking during the D12,then slightly decrease at D18(P <0.05) which decline quicker than the change of blood progesterone changes at pregnant D18.Conclusion:1. During mice pregnancy, the proportion of CD4+CD25+Foxp3+Treg and its Foxp3expression from spleen and deciduas was correlated with the changes of blood concentration of progesterone, suggesting progesterone may play a role in regulating the Treg cells in immune tolerance during pregnancy.2. The proportion of CD4+CD25+Foxp3+Treg from deciduas was higher than that from spleen, suggesting the importance of deciduas immune tolerance. Chapter ⅡThe role and mechanism of progesterone on CD4+CD25+Foxp3+regulatory T cells during pregnancyObjective:By constructing the model of ovariectomy mice (OVX), we explored the role of progesterone and its specific antagonist RU486on proportion of CD4+CD25+Foxp3+Treg, Foxp3expression, positive rate of Galectin-1on Treg and its cytokine secretion.Methods:By constructing the model of ovariectomy mice (OVX), physiological level of progesterone was injected to mice as the early pregnant state. Flow cytometry were used to detect the proportion of CD4+CD25+Foxp3+Treg in CD4+T cells and positive rate of Galectin-1on Treg surface from decidua. RT-PCR was used to detect Foxp3mRNA expression, ELISA was used to detect the level of IL-2、 IL-10and IFN-γ from deciduas.Results:1. In OVX+P4and OVX+P8groups, the proportion of CD4+CD25+Foxp3+Treg, its Foxp3mRNA expression level and positive rate of Galectin-1on Treg surface from decidua increase depending on progesterone amount, Th2cytokines like IL-10increase and Thl cytokines like IL-2and IFN-ydecrease, in control with OVX group and OVX+Oil group (P<0.05)2. If RU486was given to OVX+P group, the increase of proportion of CD4+CD25+Foxp3+Treg, its Foxp3mRNA expression level and positive rate of Galectin-1on Treg surface from decidua will disappear., Th2cytokines like IL-10decrease and Thl cytokines like IL-2and IFN-yincrease, in control with OVX group and OVX+Oil group (P<0.05)3. Different dose of RU486was given to normal pregnant mice, the proportion of CD4+CD25+Foxp3+Treg, its Foxp3mRNA expression level and positive rate of Galectin-1on Treg surface from decidua decrease depending on progesterone amount, Th2cytokines like IL-10decrease and Thl cytokines like IL-2and IFN-yincrease, in control with OVX group and OVX+Oil group (P<0.05)Conclusion:1. Physiologic dose of progesterone could increase the proportion of CD4+CD25+Foxp3+Treg and its Foxp3expression from deciduas which in consistent with level of progesterone.2. Progesterone may increase the Treg by the pathway of progesterone receptor on Treg, increasing Galectin-1expression and Th2cytokines secretion, maintaining the immune tolerance in pregnancy.There are12figures,47references. Chapter IIIThe role of progesterone on CD4+CD25highFoxp3+regulatory T cells in human early stage of pregnancyObjective:We explored the change of proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression before and after therapy of progesterone injection for threatened abortion. And RU486was used for medical abortion, changes of proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression was investigated.Methods:1. The change of proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression before and after therapy of progesterone injection for threatened abortion.2. RU486was used for medical abortion, changes of proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression was investigated.Results:1. In normal pregnancy and early threatened abortion groups, the blood progesterone level, proportion of CD4+CD25highFoxp3+Treg and its Foxp3mRNA expression level was higher than nonpregnant group (P<0.05), early threatened abortion group was lower than normal pregnant group (P<0.05)2. The blood progesterone level, proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression was higher after progesterone therapy for threatened abortion in contrast with that before therapy (P<0.05)3. The blood progesterone level, proportion of CD4+CD25highFoxp3+Treg and its Foxp3expression was higher in artificial abortion group than medical abortion group using RU486(P<0.05) Conclusion:1. Threatened abortion patient with lower progesterone level have low proportion of CD4+CD25highFoxp3+Treg and impaired Foxp3expression suggesting Treg plays an important role in normal human pregnancy.2. Proportion of CD4+CD25highFoxp3+Treg and Foxp3expression was increase by progesterone therapy in threatened abortion patients.3. RU486may decrease the proportion of CD4+CD25highFoxp3+Treg and Foxp3expression in deciduas, suggesting progesterone regulates Treg by progesterone receptor.