调节性T细胞在母胎耐受中的作用机制及对H-Y皮肤移植影响的研究

发布时间：2018-02-12 05:56

  本文关键词： 雌激素 孕激素 调节性T细胞 性别 小鼠 母胎耐受 调节性T细胞 妊娠 抗原特异性 小鼠 雌激素 孕激素 调节性T细胞 H-Y抗原 小鼠　出处：《华中科技大学》2010年博士论文　论文类型：学位论文

【摘要】： 第一部分妊娠水平的雌、孕激素对机体免疫系统的影响及机制研究 目的探讨妊娠浓度雌、孕激素对小鼠体内调节性T细胞的影响及可能机制。 方法实验分为体内部分和体外部分。体内实验：建立去势C57BL／6小鼠模型,两周后分别予以皮下注射雌激素(E2)、孕激素(P4)、雌孕激素联合及溶剂(生理盐水)模拟重建妊娠浓度小鼠激素水平。维持两周后,流式细胞术和免疫组化检测给药后小鼠外周血、脾脏、腹腔淋巴结、胸腺中调节性T细胞(Treg)比例变化情况。并通过ELISA的方法检测外周血中TNF-α、IL-2、IFN-γ、IL-10、TGF-β等细胞因子水平的变化。体外实验：通过CFSE标记磁珠分选的调节性T细胞反应细胞,外加E2和共刺激信号,观察标记的调节性T细胞是否发生增殖,并进一步通过CFSE标记法结合单向混合淋巴细胞培养来验证其调节性T细胞的功能。 结果体内实验：无论雌、雄小鼠,去势后单独的妊娠水平的E2即可明显提高外周血、脾脏、腹腔淋巴结中CD4+CD25+Foxp3+Treg占CD4+T细胞的比例(分别为6.13±1.32%；10.50±1.85%；7.63±1.68%),与溶剂组相比有明显统计学差异(P0.05)。但在胸腺中CD4+CD25+Foxp3+Treg的比例与溶剂组没有统计学差异。与此同时,单独的妊娠水平的P4相比于溶剂组,Treg比例有所升高,但不具有统计学意义(P0.05)。而联合应用雌、孕激素对Treg的影响与单独应用E2的结果类似,两者没有统计学差异(P0.05)。相同干预药物,Treg的比例变化在雌雄两组之间没有差异(P0.05)。同样的结果在免疫组化中得到验证。对于外周血中细胞因子的影响,在没有外来抗原刺激的条件下是没有多大影响的。 体外实验：单独的E2处理对于调节性T细胞并不能直接诱导CFSE标记的调节性T细胞的增殖(PI=1.01±0.01),单独的TCR双信号刺激也不能检测到CFSE的增殖(PI=1.03±0.02),只有在E2的预处理联合CD3/CD28的刺激下才能检测到调节性T细胞自身的增殖(PI=1.15±0.03)。验证了E2对于调节性T细胞数量上的促增殖作用之后,功能学方面的实验结果提示,CD3／CD28的刺激能显著促进CD4+CD25- NaiveT细胞的增殖(PI=1.56±0.03),在此基础上按照反应细胞与调节性T细胞比为1：1,未经E2处理组也能检测到对刺激后增殖的抑制(PI=1.18±0.04),E2的预处理能进一步增强调节性T细胞的抑制功能(PI=1.07±0.02),不仅与刺激组相比有差异(1.07±0.02 vs 1.56±0.03,P0.05),与未经E2处理组同样有差异(1.07±0.02 vs 1.18±0.04,P0.05)证明了E2不仅能通过促进调节性T细胞的增殖而扩增,而且在功能上也能进一步增强其对效应T细胞活化后增殖的抑制效应。 结论单独应用妊娠浓度E2可明显提高小鼠外周血,脾脏,腹腔淋巴结CD4+CD25+Foxp3+Treg比例,妊娠浓度的P4对外周Treg没有影响，，两种激素对中枢淋巴器官的淋巴细胞均没有影响。这可能与小鼠T淋巴细胞中雌、孕激素的受体分布、功能及与性别遗传没有相关性有关。体外实验的结果不仅验证妊娠水平的E2通过改变调节性T细胞的无反应性,在联合TCR信号下能促进调节性T细胞自身发生增殖,还能在功能上加强调节性T细胞的抑制作用。无论是中枢还是外周的调节性T细胞的比例均没有影响.妊娠水平的雌、孕激素对于血清中细胞因子免疫网络是没有太大的影响。 第二部分调节性T细胞在妊娠保护中的作用及抗原特异性的研究 目的：探讨妊娠中调节性T细胞的免疫调节作用是否具有抗原特异性。 方法：本实验分体外部分和体内部分。体外实验：以雌性CBA/J与雄性DBA/2J同笼构建流产趋势模型,从流产趋势孕10天雌鼠脾脏分离的单个核细胞经尼龙毛处理后作为效应T细胞(Tef)；DBA雄鼠脾细胞经MMC处理后作为APC；根据培养体系中调节性T细胞(Treg)的来源分组：未孕CBA雌鼠来源的Treg为未孕组,正常妊娠(CBA/J×Balb/c)来源的Treg为正常妊娠组,无关正常妊娠(Balb/c×C57BL/6)来源的Treg为正常妊娠无关对照组。按照Tef与Treg比分别为10：1,1：1,1：5构建单向混合淋巴细胞培养体系,72小时后,流式细胞仪检测标记细胞的增殖指数(Proliferative Index, PI), ELISA检测细胞因子水平。 体内实验：构建流产趋势的妊娠模型,于妊娠第0-2天,过继输注来自正常妊娠组、未孕组、无关妊娠对照组的CD4+CD25+Treg,观察Treg的过继输注对妊娠结局的影响。按照Treg的来源不同分为：正常妊娠组、未孕组、无关妊娠对照组。 结果：体外实验：在Tef与Treg比例为10：1的体系中,未孕组效应性T细胞增殖明显受到抑制。在1：1,1：5的体系中同样均能检测到明显的抑制效应,但这种抑制效应没有剂量依赖性。而分别来自于其他两组正常妊娠小鼠的Treg也表现出相似的增殖抑制效应,各组之间没有统计学差异。在Treg的干预后培养体系中细胞因子水平的变化在Treg的干预后,IL-10的水平明显升高,并且这种水平的升高呈Treg剂量依赖性正相关。而IFN-γ的水平明显减少,并且与Treg剂量呈负相关。 体内实验：在外周血中,流产趋势组CD4+CD25+Foxp3+Treg细胞占CD4+T细胞的比例较未孕组明显增高(6.59% vs 3.55%,P0.05)；正常妊娠组中外周血能检测到更为显著的Treg的升高,不仅与未孕组有差异(8.34% vs 3.55%,P0.05),与流产趋势组亦有明显差异(8.34% vs 6.59%,P0.05)。同样,在淋巴结(6.86% vs 4.85；9.62%vs 4.85%, P0.05; 9.62% vs 6.86%, P0.05)中也获得相似结果。过继输注后,只有来自正常妊娠组小鼠的Treg的过继输注才能完全阻止流产的发生。而来自未孕组和无关对照组的Treg不能逆转流产的发生。Treg过继输注不能改变体内Th1型细胞因子水平,但是能显著提升IL-10水平。 结论：无论哪种来源的Treg都具有免疫调节的潜能。在体外均能检测到对Treg增殖的抑制效应,但并没有表现出抗原特异性的更为显著抑制功能；Treg在体外可能是通过分泌高浓度的IL-10发挥抑制效应。但在体内正常的妊娠过程中产生的Treg在保护胚胎免受母体攻击方面是具有抗原特异性的,只有之前接触胎儿父系抗原的具有抗原特异性的Treg才能在体内发挥对胎儿的保护作用。 第三部分妊娠水平的雌、孕激素在H-Y皮肤移植中的作用及其机制研究 目的探讨妊娠浓度雌激素(E2)、孕激素(P4)对小鼠H-Y皮肤移植的影响及其机制。 方法建立小鼠去势模型,每日分别注射E2、P4及E2+P4联合注射,14d后受鼠行H-Y皮肤移植并继续给药。检测移植前及移植后第3天外周血中CD4+CD25+Foxp3+调节性T细胞(Treg)变化及外周血细胞因子水平；观察H-Y皮肤移植物的存活。 结果E2可提高外周血Treg的比例(6.13±1.32% vs 3.26±0.68%,P0.05),移植后Treg的比例进一步升高(11.78±2.32% vs 3.33±0.57%,P0.05)；P4对Treg没有显著影响(4.01±0.79% vs 3.26±0.68%,P0.05),而移植前后E2+P4联合对Treg的影响与单独应用E2结果相似(6.13±1.32% vs 7.25±1.71%,P0.05;12.35±3.04% vs 11.78±2.32%,P0.05)。移植后P4对Treg仍无明显影响,而E2+P4联合亦不能进一步提高Treg的比例。细胞因子检测显示,E2、P4均能减少促炎因子分泌,并提高抗炎因子分泌(P0.05)。E2、P4均能有效延长H-Y皮肤移植物存活((44.0±1.2天,35.5±0.8天,23.0±1.6天,P0.05,P0.05),且两者联用具有协同效应(MST=56.5±3.2天)。结论P4可通过诱导细胞因子免疫偏移促进H-Y皮肤移植物的存活。而E2除诱导细胞因子免疫偏移外,还通过提高Treg水平而延长移植物的存活。
[Abstract]:The effect of estrogen and progesterone on the immune system in the first part of pregnancy and its mechanism
Objective to investigate the effect of pregnancy hormone and progesterone on regulatory T cells in mice and its possible mechanism.
Methods the experiment was divided into in vivo and in vitro. In vivo experiment: to establish ovariectomized C57BL / 6 mice model, two weeks after subcutaneous injection of estrogen respectively (E2), progesterone, estrogen and progesterone (P4) and solvent (saline) simulated reconstruction of hormone levels. Pregnancy concentrations in mice maintained after two weeks, flow cytometry operation and immunohistochemical detection of mice after administration of peripheral blood, spleen, lymph node, regulatory T cells in the thymus (Treg) the change of proportion. And TNF- alpha, detected by ELISA method in peripheral blood IL-2, IFN- gamma, IL-10, TGF- and other factors and levels of beta cells in vitro. By CFSE marker sorting of regulatory T cells reaction cells with E2 and costimulatory signals, whether T cell proliferation regulation observation marker, and further by CFSE labeling and one-way mixed lymphocyte culture to verify the regulatory T cell function.
The results of experiment in vivo: both female and male mice after castration alone pregnancy can significantly improve the level of E2 in peripheral blood, spleen, CD4+CD25+Foxp3+Treg as a percentage of CD4+T cells in the abdominal lymph node (6.13 + 1.32%; 10.50 + 1.85%; 7.63 + 1.68%), there was a significant difference compared with the solvent group (P0.05) CD4+CD25+Foxp3+Treg. But in the thymus and the proportion of solvent group was not statistically significant. At the same time, single pregnancy levels of P4 compared to the solvent group, the proportion of Treg increased, but not statistically significant (P0.05). And the combination of estrogen, effect of progesterone on Treg with E2 alone was similar, there was a significant difference between the two (P0.05). The same drug intervention, the proportion of Treg changes in the male and female did not differ between the two groups (P0.05). The same result was verified in immunohistochemistry. The effect of cytokines in peripheral blood, not in There is not much effect on the condition of external antigen stimulation.
The proliferation of E2 in vitro: the separate treatment of regulatory T cells to regulatory T cells did not directly induce CFSE markers (PI=1.01 + 0.01), separate the proliferation of TCR co stimulation can not detect the CFSE (PI=1.03 + 0.02), the only treatment combined with CD3/CD28 in E2 pre stimulation can be detected regulatory T cells proliferation (PI=1.15 + 0.03). After the validation of the E2 for the proliferation of regulatory T cell number, the functional aspects of the experiment results indicated that CD3 / CD28 stimulation can significantly promote the proliferation of NaiveT cells (CD4+CD25- PI= 1.56 + 0.03), on the basis of reaction cell regulatory T cell ratio was 1:1, with no E2 treatment group was also detected on the inhibition of proliferation after stimulation (PI=1.18 + 0.04), E2 pretreatment can further enhance the suppressive function of regulatory T cells (PI=1.07 + 0.02), not only has the difference compared with the stimulation group ISO (1.07 + 0.02 vs 1.56 + 0.03, P0.05), and without E2 treatment group has the same difference (1.07 + 0.02 vs 1.18 + 0.04, P0.05) show that E2 can not only promote and amplified by regulatory T cell proliferation, but also can further enhance the inhibitory effect on the proliferation of T cell activation effect after the function.
Conclusion single application can significantly increase the concentration of E2 in peripheral blood, spleen CD4+CD25+Foxp3+Treg ratio of pregnancy, abdominal lymph nodes, pregnancy did not affect the concentration of P4 in peripheral Treg lymphocytes, there is no effect of two hormones on central lymphoid organs. This may be related to mouse T cells in female, progesterone receptor distribution, function and there is no correlation between genetic sex. In vitro results not only validate the pregnancy level of E2 by changing the response of regulatory T cells, can promote the proliferation of regulatory T cells in combination with TCR signal, and can strengthen the inhibitory effect of regulatory T cells on the function. Whether there is no effect of the proportion of regulatory T the central or peripheral cells. The level of female pregnancy, progesterone is not much impact on serum cytokine immune network.
The role of the second part of regulatory T cells in the protection of pregnancy and the study of the antigen specificity
Objective: To investigate whether the immunoregulatory effect of regulatory T cells in pregnancy has antigenic specificity.
Methods: this experiment in vitro and in vivo. The in vitro part: female CBA/J and male DBA/2J with cage construction abortion trend model, from the mononuclear cells of female rats at 10 days gestation abortion trend of spleen separated by the nylon wool treated as effector T cells (Tef); the spleen cells of DBA mice treated by MMC as APC; according to the development of regulatory T cells in the system (Treg) source packet: not pregnant female CBA mice Treg from non pregnant group, normal pregnancy (CBA/J * Balb/c) Treg from normal pregnant group, independent of normal pregnancy (Balb/c * C57BL/6) Treg from unrelated normal pregnancy control group. According to the Tef and Treg ratio were 10:1,1:1,1:5 to construct one-way mixed lymphocyte culture system, 72 hours after the detection of labeled cells by flow cytometry and proliferation index (Proliferative Index, PI), the detection of cytokines ELISA level.
In vivo experiment: pregnancy model abortion trend, on the 0-2 day of pregnancy, adoptive transfer from normal pregnancy group and non pregnant group, unrelated pregnant control group CD4+CD25+Treg, observe the Treg adoptive infusion on pregnancy outcome. According to the sources of Treg were divided into: normal pregnancy group and non pregnant group. Independent of pregnancy control group.
Results: in vitro, Tef and Treg in the ratio of 10:1 in the system of non pregnant group effector T cell proliferation was significantly inhibited. In 1:1,1:5 system the same were detected significantly inhibited, but the inhibition effect is not dose dependent. But from the other two groups of normal pregnant mice Treg also showed similar inhibitory effect on proliferation, no statistically significant differences between the groups. In the Treg intervention training changes of cytokine levels in the system in the Treg intervention, the level of IL-10 was significantly increased, and the increased level of Treg was dose dependent correlation. IFN- gamma level was significantly reduced, and negative correlation with the dose of Treg.
In vivo experiment: in the peripheral blood, the trend of abortion group CD4+CD25+Foxp3+Treg cells accounted for CD4+T cells was significantly higher than that of non pregnant group (6.59% vs 3.55%, P0.05); normal pregnancy group peripheral blood can be detected more significant increase in Treg, not only has the difference with the non pregnant group (8.34% vs 3.55%, P0.05) with the trend, there was obvious difference in abortion group (8.34% vs 6.59%, P0.05). Similarly, in the lymph nodes (6.86% vs 4.85; 9.62%vs 4.85%, P0.05 9.62%; vs 6.86%, P0.05) also obtained similar results. After adoptive infusion, only from mice of normal pregnancy Treg adoptive transmission occurs to note to prevent abortion completely. And from the non pregnant group and the control group of independent Treg did not reverse the occurrence of abortion.Treg adoptive infusion can not change in the level of Th1 type cytokines, but can significantly improve the level of IL-10.
Conclusion: no matter what kind of sources of Treg have immunomodulatory potential. In vitro were detected the inhibitory effect on the proliferation of Treg, but did not show specific antigen is more significant inhibitory function; Treg in vitro may exert inhibitory effects through the secretion of high concentrations of IL-10. But in the normal body during pregnancy Treg is antigen specificity in protecting the embryo from maternal fetal contact before the attacks, only paternal antigen with antigen specific Treg can play a protective effect on the fetus in vivo.
The role of estrogen and progesterone in the third part of pregnancy and its mechanism in H-Y skin transplantation
Objective to investigate the effect of pregnancy concentration estrogen (E2) and progestin (P4) on H-Y skin transplantation in mice and its mechanism.
Methods ovariectomized mice model, daily injection of E2, P4 and 14d combined injection of E2+P4, after H-Y rat skin transplantation and to continue drug detection. Third days before and after transplantation of peripheral blood CD4+CD25+Foxp3+ regulatory T cells (Treg) cytokines in peripheral blood and observe the H-Y changes; skin graft the survival.
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