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。
Chapter I
Relationship between progesterone level and CD4+CD25+Foxp3+regulatory T cells in different stages of mice pregnancy
Objective:
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 Ⅱ
The role and mechanism of progesterone on CD4+CD25+Foxp3+regulatory T cells during pregnancy
Objective:
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 III
The role of progesterone on CD4+CD25highFoxp3+regulatory T cells in human early stage of pregnancy
Objective:
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.
引文
[1]Sasaki Y, Sakai M, Miyazaki S, et al. Decidual and peripheral blood CD4+ CD25+regulatory T cells in early pregnancy subjects and spontaneous abortion cases [J]. Mol Hum Reprod,2004,10(5):347-353.
[2]Mei S, Tan J.Chen H, et al. Changes of CD4+CD25high regulatory T cells and FOXP3 expression in unexplained recurrent spontaneous abortion patients[J].Fertil Steril,2010,94(6):2244-2247.
[3]Yang H, Qiu L, Chen Q et al. Proportional change of CD4+CD25+regulatory T cells in deciduas and peripheral blood in unexplained recurrent spontaneous abortion patients[J].Fertil Steril,2008,89(3):656-661.
[4]杨卉,林其德,陈广洁,等.原因不明复发性流产患者蜕膜Foxp3 mRNA及其蛋白的表达[J].现代妇产科进展,2006,15(4)：292-294.
[5]邱丽华,林其德.调节性T淋巴细胞与原因不明复发性流产的相关性研究[J].中华妇产科杂志,2004,39(12)：816-818.
[6]杨金英,李大金,金莉萍,等.反复自然流产患者外周血调节性T细胞Foxp3的表达[J].现代免疫学,2008,28(2)：101-103.
[7]Madazli R, Aydin S, Uludag S, et al. Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels[J]. Acta Obstet Gynecol Scand,2003,82(9):797-802.
[8]Saito S, Shiozaki A, Nakashima A, et al. The role of the immune system in preeclampsia[J]. Mol Aspects Med,2007,28(2):192-209.
[9]Matthiesen L, Berg G, Ernerudh J, et al. Immunology of preeclampsia[J]. Chem Immunol Allergy,2005,89:49-61.
[10]Romero R, Chaiworapongsa T, Espinoza J, et al. Fetal plasma MMP-9 concentrations are elevated in preterm premature rupture of the membranes[J]. Am J Obstet Gynecol,2002,187(5):1125-1130.
[11]Simhan HN, Caritis SN, Krohn MA, et al. The vaginal inflammatory milieu and the risk of early premature preterm rupture of membranes[J]. Am J Obstet Gynecol,2005,192(1):213-218.
[12]Saito S, Shima T, Nakashima A, et al. What is the role of regulatory T cells in the success of implantation and early pregnancy [J]? J Assist Reprod Genet,2007, 24:379-386.
[13]Wegmann TG, Lin H, Guilbert L, et al. Bidirectional cytokine interactions in the maternal-fetal relationship:is successful pregnancy a TH2 phenomenon?[J]. Immunol Today,1993,14(7):353-356.
[14]Wilczynski JR. Thl/Th2 cytokines balance-yin and yang of reproductive immunology[J]. Eur J Obstet Gynecol Reprod Biol,2005,122(2):136-143.
[15]Carosella ED, Rouas-Freiss N, Paul P, et al. HLA-G:a tolerance molecule from the major histocompatibility complex[J]. Immunol Today,1999,20(2):60-62.
[16]Bamberger AM, Schulte HM, Thuneke I, et al. Expression of the apoptosis-inducing Fas ligand (FasL) in human first and third trimester placenta and choriocarcinoma cells[J]. J Clin Endocrinol Metab,1997,82(9):3173-3175.
[17]Fontenot JD, Gavin MA and Rudensky AY. Foxp3 programs the development and function of CD4+CD2+regulatory T cells [J]. Nat Immunol,2003, 4(4):330-336.
[18]Hori S, Nomura T and Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3 [J]. Science,2003,299(5609): 1057-1061.
[19]Khattri R, Cox T, Yasayko SA, et al. An essential role for Scurfin in CD4+CD25+regulatory T cells[J]. Nat Immuol,2003,4(4):337-342.
[20]Picca CC and Caton AJ. The role of self-peptides in the development of CD4+CD25+regulatory T cells [J].Curr Opin Immunol,2005,17(2):131-136.
[21]Yi H, Zhen Y, Jiang L, et al. The phenotypic characterization of naturally occurring regulatory CD4+CD25+regulatory T cells [J].Cell Mol Immunol,2006,3(3): 189-195.
[22]Takahashi T, Tagami T, Yamazaki S, et al. Immunologic self-tolerance maintained by CD4+CD25+regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4[J]. J Exp Med,2000,192(2):303-310.
[23]Waterhouse P, Penninger JM, Timms E, et al. Lymphoproliferative disorders with early lethality in mice deficient in Ctla-4[J]. Science,1995,270(5238):985-988.
[24]Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor[J]. Annu Rev Immunol,2001,19:683-765.
[25]De Waal Malefyt R, Abrams J, Bennett B, et al. Interleukin-10 inhibits produced by monocytes[J]J Exp Med,1991,174(5):1209-1220.
[26]Bogdan C, Vodovotz Y and Nathan C. Macrophage deactivation by interleukin 10[J]. J Exp Med,1991,174(6):1549-1555.
[27]Somerset DA, Zheng Y, KIlby MD, et al. Normal human pregnancy is associated with an elevation in the immune suppressive CD25+CD4+regulatory T-cell subse [J]. Immunology,2004,112(1):38-43.
[28]Sakaguchi S. Nutrally arising Foxp3-expressing CD25+CD4+regulatory T cells in immunological tolerance to self and non-self [J]. Nat Immunol,2005, 6(4):345-352.
[29]李兴逛,高学敏.瞿麦水煎液对小鼠妊娠影响的实验研究[J].北京中医药大学学报,2000,23(6):235-238.
[30]张卫社,,谢庆生,伍招娣,等.NMBR在不同妊娠阶段小鼠子宫平滑肌细胞中的表达及其与临产的关系[J].中南大学学报(医学版),2009,34(6)：531-536.
[31]孙奎,梅丹,姜华,等.实验鼠采血方法及特点[J].第四军医大学吉林军医学院学报,2003,25(1)：31-32.
[32]林星光,汪理,高英等.孕早期水平雌孕激素对小鼠体内调节性T细胞的影响[J].医药导报,2010,7(29)：839-846.
[33]Moriyama I, Sugawa T. Progesterone facilitates implantation of xenogeneic cultured cells in hamster uterus[J]. Nat New Biol,1972,236:150-152.
[34]Padykula HA, Jansey TR. The occurrence of uterine stromal and intraepithelial monocytes and heterophils during nomal late pregnancy in the rat[J]. Anat Rec,1979,143:329-355.
[35]Louis E, Clemens, Pentti K, et al. Mechanism of immune-suppression of progesterone on maternal lymphocyte activation during pregnancy[J]. J Immunol, 1979,122:1978-1985.
[36]Bullock DW. Progesterone induction of messenger RNA and protein synthesis in rabbit uterus[J]. Ann Ny Acad Sci USA,1977,286:260-272.
[37]Pijnenborg R, Bland JM, Robertson WB, et al. Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy [J]. Placenta,1983,4(4):397-413.
[38]Wafula PO, Teles A, Schumacher A, et al. PD-1 but not CTLA-4 Blockage Abrogates the Protective Effect of Regulatory T Cells in a Pregnancy Murine Model[J]. Am J Reprod Immunol 2009,62(5):283-292.
[39]Zenclussen AC. CD4+CD25+regulatory T cells in murine pregnancy[J]. J Reprod Immunol,2005,65(2):101-110.
[40]Brunkow ME, Jeffery EW, Hjerrild KA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse [J]. Nat Genet,2001,27(1):68-73.
[41]Bennett CL, Christie J, Ramsdell F, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3 [J]. Nat Genet,2001,27(1):20-21.
[42]Graca L. New tools to identify regulatory T cells [J]. Eur J Immunol,2005, 35(6):1678-1680.
[43]焦志军,尤海燕,陈蕾,等.流式细胞术检测表达Foxp3的CD4+CD25+调节性T细胞[J].临床检验杂志,2008,26(3)：161-163.
[44]Dimova T, Nagaeva O, Stenqvist AC,et al. Maternal Foxp3 expressing CD4+ CD25+and CD4+CD25- regulatory T-cell populations are enriched in human early normal pregnancy decidua:a phenotypic study of paired decidual and peripheral blood samples[J]. Am J Reprod Immunol.2011,7;66 Suppl 1:44-56.
[45]Ernerudh J, Berg G, Mjosberg J. Regulatory T helper cells in pregnancy and their roles in systemic versus local immune tolerance[J]. Am J Reprod Immunol. 2011,65(Suppl.1):31-43.
[46]Jin LP, Chen QY, Zhang T,et al. The CD4+CD25 bright regulatory T cells and CTLA-4 expression in peripheral and decidual lymphocytes are down-regulated in human miscarriage[J]. Clin Immunol.200912;133(3):402-410.
[47]Perillo NL,Pace KE, Seilhamer JJ, et al. Apoptosis of T cells mediated by galectin-1. Nature,1995,378(6558):736-9.
[48]冯婷婷,郭俊成,刘岳婷,等.不明原因复发性流产小鼠模型CD4+CD25+Treg的比例及Foxp3表达的变化[J].免疫学杂志.2012,7(28)：595-600.
[49]Mao G, Wang J, Kang Y, et al. Progesterone increases systemic and local uterine proportions of CD4+CD25+Treg cells during midterm pregnancy in mice. Endocrinology.2010.11;151(11):5477-5488.
[50]潘卫,王应雄,黎刚,等.小鼠胚泡薪附时子宫内膜Galectin-1的变化[J].生殖与避孕,2004,24(3)：129-3.
[51]Maldonado CA. Regulated expression and ultrastructural localization of galectin-1, aproapoptotic beta-galactoside-binding lectin, during spermatogenesis in rat testis.Biol Reprod,2003,68(1):51-59.
[52]孔紫靖,胡丽芳,李允光,等.Galectin-1基因在人孕早期绒毛和蜕膜中表达的研究[J].生殖与避孕.2007.1(27)：38-41.
[53]Perillo NL,Pace KE, Seilhamer JJ, et al. Apoptosis of T cells mediated by galectin-1. Nature,1995,378(6558):736-9.
[54]Perillo NL, Uittenboqaart CH, Nquyen JT, et al. Galectin-1, an endogenous lectin produced by thymic epithelial cells, induces apoptosis of human thymocytes. J exp Med,1997,185(10):1851-8.
[55]Marina GI, Chu CC, Golshayan D,et al. Galectin-1:a key effector of regulation mediated by CD4+CD25+T cells[J]. Blood.2007.109:2058-2065.
[56]He J, Baum LG. Presentation of galectin-1 by extracellular matrix triggers T cell death. J Biol Chem.2004;279:4705-4712.
[57]Pace KE, Lee C, Stewart PL,et al. Restricted receptor segregation into membrane microdomains occurs on human T cells during apoptosis induced by galectin-1. J Immunol.1999;163:3801-11.
[58]Chung CD, Patel VP,Moran M,et al. Galectin-1 induces partial TCR zeta-chain phosphorylation and antagonizes processive TCR signal transduction. J Immunol.2000; 165:3722-29.
[59]Rabinovich GA, Daly G, Dreja H, et al. Recombinant galectin-1 and its genetic delivery suppress collagen-induced arthritis via T cell apoptosis. J Exp Med. 1999; 190:385-98.
[60]Rabinovich GA, Baum LG, Tinari N, et al. Galectins and their ligands: amplifiers, silencers or tuners of the inflammatory response? Trends Immunol. 2002;23:313-320.
[61]Rabinovich GA, Ariel A, Hershkoviz R, et al. Specific inhibition of T-cell adhesion to extracellular matrix and proinflammatory cytokine secretion by human recombinant galectin-1. Immunology.1999;97:100-106.
[62]Blaser C, Kaufmann M, Muller C, et al. Beta-galactoside-bingding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells. Eur J Immunol.1998;28:2311-2319.
[63]罗丽兰.不孕不育[M].北京：人民卫生出版社,2000：289
[64]杨卉,林其德,邱丽华,等.原因不明复发性流产主动免疫治疗前后CD4+ CD25+调节性T细胞表达频率的研究[J].中国实用妇科与产科杂志,2006,22(7)：512-514.
[65]Shevach EM. Certified professionals:CD4(+)CD25(+)suppressor T cells[J]. J Exp Med,2001; 193(11):F41-F46.
[66]Shimizu J, Yamazaki S, TakahashiT, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance[J]. Nat Inmmnol,2002; 3(2):135-142.
[67]Camara NO, Sebille F, Lechler R1. Human CD4+CD25+regulatory cells have marked and sustained effects on CD8+T cell activation[J]. Eur J Immunol, 2003; 33(12):3473-3483.
[68]Cobbold SP, Nolan KF, Graca L, et al. Regulatory T cells and dendritic cells in transplantation tolerance:molecular markers and mechanisms. Immunol Rev, 2003; 196:109-124.
[69]Wood KJ, Ushigome H, Karim M, et al, Regulatory cells in transplantation[J]. Novartis Found Symp,2003,252:177-210.
[70]Cederbom L, Hall H, Ivars F. CD4+CD25+regulatory T cells down-regulate CO-stimulatory molecules on antigen-presenting cells[J]. Eur J Immunol,2000; 30(6):1538-1543.
[71]Von Boehmer H. Mechanisms of suppression by suppressor T cells [J]. Nat Imm un o,l 2005,6(4):338-344.
[72]Kullberg, MC, D. Jankovic, et al. Bacteria-triggered CD4+T regulatory cells suppress Helicobacter hepaticus-induced colitis. J. Exp.Med.2002;196:505-515.
[73]Dieckmann D, Plottner H, Berchtold S,et al. Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J Exp Med 2001; 193:1303-1310.
[74]Jonuleit H, Schmitt E, Stassen M, et al.Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J Exp Med 2001; 193:1285-1294.
[75]Levings MK, Sangregorio R, Roncarolo MG. Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 2001; 193:1295-1302.
[76]Ng WF, Duggan PJ, Ponchel F, Matarese G, et al. Human CD4(+)CD25(+) cells:a naturally occurring population of regulatory T cells. Blood 2001; 98:2736-2744.
[77]Taams LS, Smith J, Rustin MH, et al. Human anergic/suppressive CD4(+)CD25(+) T cells:a highly differentiated and apoptosis-prone population. Eur J Immunol 2001;31:1122-1131.
[78]Maloy KJ, Salaun L, Cahill R, et al. CD4+CD25+T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. J Exp Med 2003; 197:111-119.
[79]Vignali DA, Collison LW, Workman CJ. How regulatory T cells work. Nat Rev Immunol 2008; 8:523-532.
[80]Collison LW, Pillai MR, Chaturvedi V, et al. Regulatory T cell suppression is potentiated by target T cells in a cell contact, IL-35-and IL-10-dependent manner. J Immunol 2009; 182:6121-6128.
[81]Collison LW, Workman CJ, Kuo TT, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007; 450:566-569.
[82]Bardel E, Larousserie F, Charlot-Rabiega P, et al. Human CD4+CD25+ Foxp3+regulatory T cells do not constitutively express IL-35. J Immunol 2008; 181:6898-6905.
[83]Oida T, Xu L, Weiner HL, Kitani A, et al. TGF-beta-Mediated Suppression by CD4+CD25+T cells is Facilitated by CTLA-4 signaling. J Immunol. 2006.177(4):2331-9.
[84]Grohmann U, Orabona C, Fallarino F, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002;3:1097-101.
[85]Fallarino F, Grohmann U, Hwang KW, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol 2003;4:1206-12.
[86]Munn DH, Zhou M, Attwood JT, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998;281:1191-3.
[87]Munn DH, Shafizadeh E, Attwood JT, et al. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 1999; 189:1363-72.
[88]Munn DH, Sharma MD, Lee JR, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002;297:1867-70.
[89]Miwa N, Hayakawa S, Miyazaki S, et al. IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion. Mol Hum Reprod 2005;11:865-70.
[90]Corthay A.How do regulatory T cells work? Scand J Immunol 2009; 70:326-336.
[1]Saito S, Nishikawa K, Morii T, Narita N, Enomoto M, Ichijo M.Expression of activation antigens CD69, HLA-DR, interleukin-2 receptor-alpha (IL-2Ra) and IL-2Rβ on T cells of human deciduas at an early stage of pregnancy. Immunology 1992;75:710-2.
[2]Baecher-Allan C, Brown JA, Freeman GJ, et al. CD4+CD25high regulatory cells in human peripheral blood. J Immunol,2001;167:1245-53.
[3]Baecher-Allan C, Brown JA, Freeman GJ, et al. CD4+CD25high regulatory cells from human peripheral blood express very high levels of CD25 ex vivo. Novartis Found Symp,2003;252:67-88.
[4]Sakaguchi,S. Naturally arising CD4+regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev Immunol.2004.22:531-62.
[5]Schevach, EM. CD4+CD25+suppressor T cells:more questions than answers. Nat. Rev. Immunol.2002,2:389-400.
[6]Kullberg, M>C>, D. Jankovic, et al. Sher. Bacteria-triggered CD4+T regulatory cells suppress Helicobacter hepaticus-induced colitis. J. Exp.Med. 2002; 196:505-515.
[7]Belkand, Y, CA Piccirillo, S Mendez, et al. CD4+CD25+regulatory T cells control Leishmania major persistence and immunity. Nature.2002;420:502-507
[8]Trenado A, F Charlotte, S Fisson, et al. Recipient-type specific CD4+ CD25+regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia. J Clin Invest.2003.112:1688-96.
[9]Shimizu J, S Yamazaki, S Sakaguchi. Induction of tumor immunity by removing CD4+CD25+T cells:a common basis between tumor immunity and autoimmunity. J Immunol.1999.163:5211-8.
[10]Onizuka S, I Tawara, J Shimizu, et al. Tumor rejection by in vivo administration of anti-CD25(interleukin-2 receptor a) monoclonal antibody. Cancer Res.1999.59:3128-33.
[11]Steitz J, Bruck J, Lenz, et al. Depletion of CD4+CD25+T cells and treatment with tyrosinase-related protein 2-transduced dendric cells enhance the interferon a-induced CD8+T-cell-dependent immune defense of B16 melanoma. Cancer Res.2001.61:8643-6.
[12]Turk MJ, JA Guevara-Patino, et al. Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells.J Exp Med. 2004.200:771-82.
[13]Pai SY, Truitt ML, Ting CN, et al. Critical roles for transcription factor GATA-3 in thymocyte development[J]Immunity,2003; 19(6):863-875.
[14]Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3.Science,2003; 299(5609):1057-1061.
[15]Kim HJ, Kim HY, Kim BK, et al. Engagement of glucocorticoid-induced TNF receptor costimulates NKT cell activation in vitro and in vivo. J Immunol.2006.176(6):3507-15.
[16]Read S, V Malmstrom, F Powrie. Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD4+CD25+regulatory cells that control intestinal inflammation. J Exp Med.2000.192:295.
[17]Wei L, Wei-Min L, Cheng G, et al. Upregulation of CD4+CD25+T lymphocyte by adenovirus-mediated gene transfer of CTLA4 Ig fusion protein in experimental autoimmune myocarditis. Autoimmunity.2006.39(4):289-98.
[18]Shevach EM. Certified professionals:CD4(+)CD25(+)suppressor T cells. J Exp Med,2001; 193(11):F41-F46.
[19]Shimizu J, Yamazaki S, Takahashi T, et al. Stimulation of CD25(+)CD4(+) regulatory T cells through GITR breaks immunological self-tolerance. Nat Inmmnol, 2002; 3(2):135-142.
[20]Camara NO, Sebille F, Lechler R1. Human CD4+CD25+regulatory cells have marked and sustained effects on CD8+T cell activation[J]. Eur J Immunol, 2003; 33(12):3473-3483.
[21]Cobbold SP, Nolan KF, Graca L, et al. Regulatory T cells and dendritic cells in transplantation tolerance:molecular markers and mechanisms. Immunol Rev, 2003;196:109-124.
[22]Wood KJ, Ushigome H, Karim M, et al, Regulatory cells in transplantation. Novartis Found Symp,2003; 252:177-210.
[23]Cederbom L, Hall H, Ivars F. CD4+CD25+regulatory T cells down-regulate CO-stimulatory molecules on antigen-presenting cells[J]. Eur J Immunol,2000; 30(6):1538-1543.
[24]von Boehm er H. M ech an ism s of suppression by suppressor Tcells [J]. Nat Immunol 2005,6(4):338-344.
[25]Dieckmann D, Plottner H, Berchtold S, Berger T, Schuler G:Ex vivo isolation and characterization of CD4(+)CD25(+) T cells with regulatory properties from human blood. J Exp Med 2001;193:1303-1310.
[26]Jonuleit H, Schmitt E, Stassen M, Tuettenberg A, Knop J, Enk AH: Identification and functional characterization of human CD4(+)CD25(+) T cells with regulatory properties isolated from peripheral blood. J Exp Med 2001; 193:1285-1294.
[27]Levings MK, Sangregorio R, Roncarolo MG:Human cd25(+)cd4(+) t regulatory cells suppress naive and memory T cell proliferation and can be expanded in vitro without loss of function. J Exp Med 2001; 193:1295-1302.
[28]Ng WF, Duggan PJ, Ponchel F, Matarese G, Lombardi G, Edwards AD, Isaacs JD, Lechler RI:Human CD4(+)CD25(+) cells:a naturally occurring population of regulatory T cells. Blood 2001; 98:2736-2744.
[29]Taams LS, Smith J, Rustin MH, Salmon M, Poulter LW, Akbar AN:Human anergic/suppressive CD4(+)CD25(+) T cells:a highly differentiated and apoptosis-prone population. Eur J Immunol 2001;31:1122-1131.
[30]Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, Powrie F: CD4+CD25+T(R) cells suppress innate immune pathology through cytokine-dependent mechanisms. J Exp Med 2003; 197:111-119.
[31]Vignali DA, Collison LW, Workman CJ:How regulatory T cells work. Nat Rev Immunol 2008; 8:523-532.
[32]Collison LW, Pillai MR, Chaturvedi V, Vignali DA:Regulatory T cell suppression is potentiated by target T cells in a cell contact, IL-35-and IL-10-dependent manner. J Immunol 2009; 182:6121-6128.
[33]Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, Cross R, Sehy D, Blumberg RS, Vignali DA:The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007; 450:566-569.
[34]Bardel E, Larousserie F, Charlot-Rabiega P, Coulomb-L'Hermine A, Devergne O:Human CD4+CD25+Foxp3+regulatory T cells do not constitutively express IL-35. J Immunol 2008; 181:6898-6905.
[35]Oida T, Xu L, Weiner HL, Kitani A, et al. TGF-beta-Mediated Suppression by CD4+CD25+T cells is Facilitated by CTLA-4 signaling. J Immunol. 2006.177(4):2331-9.
[36]Grohmann U, Orabona C, Fallarino F, Vacca C, Calcinaro F, Falorni A, et al. CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat Immunol 2002;3:1097-101.
[37]Fallarino F, Grohmann U, Hwang KW, Orabona C, Vacca C, Bianchi R, et al. Modulation of tryptophan catabolism by regulatory T cells. Nat Immunol 2003;4:1206-12.
[38]Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998;281:1191-3.
[39]Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 1999;189:1363-72.
[40]Munn DH, Sharma MD, Lee JR, Jhaver KG, Johnson TS, Keskin DB, et al. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 2002;297:1867-70.
[41]Miwa N, Hayakawa S, Miyazaki S, Myojo S, Sasaki Y, Sakai M, et al. IDO expression on decidual and peripheral blood dendritic cells and monocytes/macrophages after treatment with CTLA-4 or interferon-gamma increase in normal pregnancy but decrease in spontaneous abortion. Mol Hum Reprod 2005; 11:865-70.
[42]Corthay A:How do regulatory T cells work? Scand J Immunol 2009; 70:326-336.
[43]Seo N, Hayakawa S, Takigawa M,et al. Interleukin-10 expressed at early tumour sites induces subsequent generation of CD4+T regulatory cells and systemic collapse of antitumour immunity. Immunology.2001.103:449-57.
[44]Skapenko A, Kalden JR, Lipsky PE, et al. The IL-4 receptor alpha-chain-binding cytokines, IL-4 and IL-13, induce forkhead box P3-expressing CD4+CD25+regulatory T cells from CD4+CD25-precursors. J Immunol. 2005.175:6107-16.
[45]Offner H, Vandenbark AA. Congruent effects of estrogen and T-cell receptor peptide therapy on regulatory T cells in EAE and MS. Int Rev Immunol. 2005.24(5-6):447-77.
[46]Polanczyk MJ, Hopke C, Huan J, et al. Enhanced FoxP3 expression and Treg cell function in pregnant and estrogen-treated mice. J Neuroimunol.2005.170 (1-2):85-92.
[47]Prieto GA, Rosenstein Y:Oestradiol potentiates the suppressive function of human CD4 CD25 regulatory T cells by promoting their proliferation. Immunology 2006; 118:58-65.
[48]Fraccaroli L, Alfieri J, Larocca L, Calafat M, Mor G, Leiros CP, Ramhorst R:A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation. Hum Reprod 2009; 24:166-175.
[49]Garin MI, Chu C-C, Golshayan D, Cernuda-Morollon E, Wait R, Lechler RI. Galectin-1:a key effector of regulation mediated by CD4+CD25+ T cells. Blood 2007;109:2058-65.
[50]Dosiou C, Giudice LC. Natural killer cells in pregnancy and recurrent pregnancy loss:endocrine and immunologic perspectives. Endocr Rev 2005;26:44-62.
[51]Aluvihare VR, Kallikourdis M, Betz AG. Regulatory T cells mediate maternal tolerance to the fetus. Nat Immunol 2004;5:266-71.
[52]Zenclussen AC, Gerlof K, Zenclussen ML, Sollwedel A, Bertoja AZ, Ritter T, et al. Abnormal T-cell reactivity against paternal antigens in spontaneous abortion: adoptive transfer of pregnancy induced CD4+CD25+T regulatory cells prevents fetal rejection in a murine abortion model. Am J Pathol 2005; 166:811-22.
[53]Sasaki Y, Msakai S, Miyazaki et al. Decidual and peripheral blood CD4+ CD25+regulatory T cells in early pregnancy subjects and spontaneous abortion cases[J]. Molecular Human Reproduction,2004,10(5):347-353.
[54]Somerset DA, Zheng Y, Kilby MD, Sansom DM, Drayson MT. Normal human pregnancy is associated with an elevation in the immune suppressive CD4+CD25+regulatory T-cell subset. Immunology 2004; 112:38-43.
[55]Heikkinen J, Mottonen M, Alanen A, Lassila O. Phenotype characterization of regulatory T cells in the human decidua. Clin Exp Immunol 2004;136:373-8.
[56]Hui Yang, et a.l Proportional change of CD4+CD25+regulatory T cells in unexplained recurrent spontaneous abortion patients[J]. Fert ility and Sterility,2007.
[57]Ana C, zenclussen, Katrin Gerlof, Maria L, Zenclussen, et al. Regu latory T cells induce a privileged tolerant microenvironmen t at the fetal-maternal interface[J]. Eu r J Immunol.2006,36:82-94.
[58]Cosmi L, Liotta F, Lazzeri E, et al. Human CD8+CD25+thym ocytes share phenotypic and function al features with CD4+CD25+regulatory thymotytes. Blood, 2003,102 (12):4107-4114.
[59]杨卉,林其德,肖云山,等.Foxp3在子宫内膜、正常早孕和原因不明复发性流产患者蜕膜上的表达[J].现代妇产科进展,2005,14(2)：130-131.