【作者】 马敬；

【导师】 周昌菊；

【作者基本信息】 中南大学 ， 临床医学， 2013， 博士

【摘要】 第一章小鼠妊娠各时期孕酮与CD4+CD25+Foxp3+调节性T细胞的关系目的：通过观察昆明小鼠妊娠不同时期的外周血孕酮浓度、脾和子宫蜕膜处CD4+CD25+Foxp3+调节性T细胞(Regulatory T cell, Treg)含量以及Foxp3的mRNA和蛋白表达的变化规律,探讨妊娠期孕酮水平与外周淋巴组织中和子宫蜕膜处CD4+CD25+Foxp3+Treg细胞数目和功能间的关系。方法：构建昆明小鼠正常妊娠模型,雌雄2：1合笼饲养,每日晨检查雌鼠阴道栓,见栓日计为孕0天,每6只相同孕龄的小鼠作为一组,共6组,分别于孕0、4、8、12、18天和分娩后一小时颈椎脱臼法处死小鼠,健康未孕雌鼠6只作为对照组,各组取外周血通过酶联免疫吸附法(enzyme-linked immuno sorbent assay, ELISA)检测孕酮浓度,取脾脏和子宫蜕膜组织制备单细胞悬液,应用流式细胞仪分别检测脾和子宫蜕膜处CD4+CD25+Foxp3+Treg细胞的含量,反转录酶-聚合酶链锁反应法(reverse transcription-polymerase chain reaction, RT-PCR)检测Foxp3的mRNA表达水平,蛋白质印迹法(Western Blot)检测Foxp3的蛋白表达水平。结果：1.小鼠外周血孕酮水平在妊娠各时期的变化如下,孕0天组为5.89±1.45ng/ml,较未孕组3.69±0.02ng/ml无统计学差异(P>0.05),孕4天孕酮水平才开始升高,达20.21±0.74ng/ml；并且随孕龄增长孕酮水平逐渐上升,孕8天组达32.00±1.15ng/ml；孕中、晚期(孕12天、18天)达高峰并维持,分别为37.84±2.57ng/ml和38.22±1.27ng/ml；分娩后孕酮水平立即下降至6.26±1.09ng/ml,较孕18天组有统计学差异(P<0.05)。2.小鼠外周淋巴组织(脾脏)中CD4+CD25+Foxp3+Treg细胞占总CD4+T细胞的比例在妊娠各时期的变化如下,孕0天Treg占4.04±0.95%,较未孕组3.97±0.13%无统计学差异(P>0.05)；孕4天Treg数量开始升高,达9.58±1.09%,并且随孕龄增长Treg数量逐渐增多,孕8天组达13.44±1.17%,孕中、晚期(孕12天、18天)达高峰并维持,分别为15.66±1.93%和15.44±0.51%,除孕0天组外孕期各组水平均高于未孕组,有统计学差异(P<0.05)；分娩后Treg数量稍下降,为15.00±1.17%,较孕18天组无统计学差异(P>0.05)。同时,Treg细胞的活性指标Foxp3的mRNA表达水平,在妊娠各期与Treg含量变化一致,但分娩后Foxp3表达水平立即下降,较孕18天组差异有统计学意义(P<0.05)。可见,脾脏Treg数量及其Foxp3mRNA的表达水平与外周血孕酮水平在妊娠各期变化一致,分娩后孕酮水平和Foxp3mRNA表达均立即下降。3.小鼠子宫蜕膜处CD4+CD25+Foxp3+Treg细胞占总CD4+T细胞的比例在妊娠各时期的变化如下,Treg数量从孕0天开始升高占10.25±0.99%,并随孕周增加Treg数量逐渐增多,孕4天、孕8天分别为20.14±3.57%和29.80±0.43%,孕中期(孕12天)达高峰35.18±1.92%,孕晚期(孕18天)稍下降至28.64±3.67%,妊娠期各组水平均高于未孕组水平,差异有统计学意义(P<0.05)；分娩后蜕膜处Treg数量立即下降,较孕18天组有统计学差异(P<0.05)。同时,蜕膜处Foxp3的mRNA和蛋白表达水平在妊娠各期及分娩后与Treg含量变化均一致。可见,蜕膜Treg含量及其Foxp3mRNA和蛋白的表达水平在妊娠各期变化与外周血孕酮变化基本一致,但在孕晚期(孕18天组)先于孕酮水平下降。结论：1.小鼠妊娠不同时期脾脏和子宫蜕膜处CD4+CD25+Foxp3+Treg细胞含量及其Foxp3表达水平与外周血孕酮变化相关,提示孕酮可能通过调控Treg细胞而维持妊娠免疫耐受。2.孕鼠CD4+CD25+Foxp3+Treg细胞在子宫蜕膜处的变化与外周血一致,且细胞比例高于外周血,提示可通过外周血Treg细胞的测定反映蜕膜处Treg细胞的变化。第二章小鼠妊娠早期孕酮对CD4+CD25+Foxp3+调节性T细胞的影响及其调控机制目的：通过构建小鼠双侧卵巢切除模型(Ovariectomy, OVX),研究孕酮及其特异性拮抗剂RU486对小鼠子宫蜕膜处CD4+CD25+Foxp3+Treg细胞含量、Foxp3mRNA表达水平和Treg细胞表面Galectin-1阳性率及其分泌细胞因子的影响。方法：构建小鼠OVX模型,皮下注射孕酮使之模拟达到孕早期外周血孕酮的生理水平,注药24小时后处死小鼠取子宫蜕膜组织制备单细胞悬液,应用流式细胞计数分析各组CD4+CD25+Foxp3+Treg细胞的数量和Treg细胞表达Galectin-1的阳性率,RT-PCR法检测Foxp3mRNA表达水平,应用ELISA检测各组IL-2、IL-10和IFN-γ的表达水平。给予正常妊娠小鼠不同浓度孕酮特异拮抗剂RU486,观察孕酮拮抗后对以上指标的影响。结果：1.给予OVX雌鼠皮下注射孕酮达到孕4天外周血孕酮的生理水平(OVX+P4组)和孕8天生理水平(OVX+P8组)的试验组中,小鼠子宫蜕膜CD4+CD25+Foxp3+Treg细胞占总CD4+T细胞的比例、蜕膜Foxp3mRNA表达水平和Treg细胞表面Galectin-1阳性率均呈剂量依赖性升高,并且母胎界面Th2型细胞因子IL-10增多,Thl型细胞因子IL-2和IFN-γ减少,较OVX组和OVX雌鼠注射同体积油剂(OVX+Oil)组差异有统计学意义(P<0.05)。2.给予OVX雌鼠孕酮前,皮下注射孕酮特异拮抗剂RU486(OVX+P+RU486组),将拮抗孕酮上调小鼠子宫蜕膜CD4+CD25+Foxp3+Treg细胞含量、Foxp3mRNA表达水平和Treg细胞表面Galectin-1阳性率的作用,同时母胎界面Th2型细胞因子IL-10减少,Th1型细胞因子IL-2和IFN-y增多,较未拮抗组(OVX+P组)差异有统计学意义(P<0.05)。3.给予正常妊娠小鼠不同剂量的孕酮特异拮抗剂RU486,小鼠子宫蜕膜CD4+CD25+Foxp3+Treg细胞含量、Foxp3mRNA表达水平和Treg细胞表面Galectin-1阳性率均呈剂量依赖性下降,并且母胎界面Th2型细胞因子IL-10减少,Thl型细胞因子IL-2和IFN-y增多,较未孕组和正常妊娠组差异有统计学意义(P<0.05)。结论：1.孕早期水平孕酮可上调小鼠子宫蜕膜CD4+CD25+Foxp3+Treg细胞数量和功能,与剂量呈正比；2.孕早期水平孕酮可上调蜕膜处CD4+CD25+Foxp3+Treg细胞表面效应分子Galectin-1表达,分泌IL-10增多,IL-2和IFN-γ减少,且该作用通过孕酮受体介导,提示孕酮调控Treg细胞功能而促进母胎免疫耐受形成。第三章孕酮在人类妊娠早期对CD4+CD25highFoxp3+调节性T细胞的作用目的：研究10例早孕先兆流产者接受黄体酮治疗前后外周血CD4+CD25highFoxp3+Treg细胞的变化和Foxp3表达的情况,再通过比较研究孕酮拮抗剂对外周血和子宫蜕膜处CD4+CD25highFoxp3+Treg细胞和Foxp3表达的影响,共同探讨孕酮对人类早期妊娠Treg细胞的影响及其调控机制。方法：1.通过对比早孕先兆流产患者黄体酮保胎治疗前后、正常同孕周妊娠者和未孕者外周血CD4+CD25highFoxp3+Treg细胞的变化和Foxp3表达的情况,探讨孕酮对人类早期妊娠的影响和对Treg细胞的作用。2.通过对比相同孕龄行药物流产(应用孕酮特异性拮抗剂RU486)者与人工流产者的外周血和子宫蜕膜CD4+CD25highFoxp3+Treg细胞和Foxp3表达的差异,探讨孕酮通过其受体调控Treg细胞变化的机制。结果：1.未孕组、正常早孕组和早孕先兆流产组患者外周血孕酮水平分别为0.46±0.22(0.19±0.88)ng/ml、31.09±7.69(20.66±42.13) ng/ml和20.81±2.26(18.34～24.54) ng/ml。正常早孕组和早孕先兆流产组均高于非孕组水平,差异有统计学意义(P<0.05);而早孕先兆流产组低于同孕周的正常早孕组水平,差异有统计学意义(P<0.05)。2.外周血单个核细胞中CD4+CD25highFoxp3+Treg细胞比例和Foxp3mRNA的表达水平,在正常早孕组和早孕先兆流产组中均高于未孕组,差异有统计学意义(P<0.05)。而早孕先兆流产组低于正常早孕组水平,差异有统计学意义(P<0.05)。与其外周血孕酮水平的变化结果一致。3.早孕先兆流产组治疗前组血孕酮水平、外周血单个核细胞中CD4+CD25highFoxp3+Treg细胞比例和Foxp3mRNA的表达水平,均低于黄体酮治疗成功组水平,高于治疗失败组水平,两组间比较差异有统计学意义(P<0.05)。4.正常早孕者药物流产治疗前外周血孕酮水平、外周血单个核细胞中CD4+CD25highFoxp3+Treg细胞比例及其Foxp3mRNA的表达水平均高于药流后水平,差异有统计学意义(P<0.05)。5.正常早孕人工流产组外周血孕酮水平、子宫蜕膜CD4+CD25highFoxp3+Treg细胞比例及其Foxp3mRNA的相对表达水平,均高于同孕周正常妊娠行RU486药物流产者,差异有统计学意义(P<0.05)。结论：1.孕早期外周血孕酮水平下降的先兆流产者,外周血CD4+CD25highFoxp3+Treg细胞数量和功能明显下降,提示Treg可能在维持人类正常妊娠中起作用。2.早期先兆流产患者用黄体酮治疗后,外周血CD4+CD25highFoxp3+Treg细胞数量和功能明显上升,提示Treg细胞水平可能通过孕酮受体受孕酮水平调节。更多还原

【Abstract】 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.更多还原