【作者】 林星光；

【导师】 陈忠华；

【作者基本信息】 华中科技大学 ， 外科学， 2010， 博士

【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(P<0.05)。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(P>0.05)。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(P>0.05)。相同干预药物,Treg的比例变化在雌雄两组之间没有差异(P>0.05)。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,P<0.05),与未经E2处理组同样有差异(1.07±0.02 vs 1.18±0.04,P<0.05)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%,P<0.05)；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%,P<0.05),与流产趋势组亦有明显差异(8.34% vs 6.59%,P<0.05)。同样,在淋巴结(6.86% vs 4.85；9.62%vs 4.85%, P<0.05; 9.62% vs 6.86%, P<0.05)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%,P<0.05),移植后Treg的比例进一步升高(11.78±2.32% vs 3.33±0.57%,P<0.05)；P4对Treg没有显著影响(4.01±0.79% vs 3.26±0.68%,P>0.05),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%,P>0.05;12.35±3.04% vs 11.78±2.32%,P>0.05)。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(P<0.05)。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,P<0.05,P<0.05),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。更多还原

【Abstract】 PartⅠEffects and Mechanisms of Pregnancy Estrogen and Progesterone on the Immune Reaction in MiceObjective To investigate whether the estrogen (E2), progesterone(P4) within the physiological concentration range of pregnancy could affect the expansion of CD4+CD25+ Foxp3+regulatory T cell. Methods In vitro, we first isolated the CD4+CD25+ regulatory T cells by magenetic columns, and then labeled the regulatory T cells with carboxyfluorescein diacetate succinimidyl ester (CFSE). We treated the labeled regulatory T cells with estrogen (E2) and anti-CD3 antibody and anti-CD28 antibody. Then we used the flow cytometry detect the changes of fluorescence, and analyse the proliferation index of the labeled cells. Furthermore, we detected the function of expanded regulatory T cells. Groups were divided as followed, Group A, labeled E2-untreated Treg with PBS; Group B, E2-treated Tregs with PBS; Group C, labeled E2-untreated Treg with CD3/CD28; Group D, labeled E2-treated Treg with CD3/.CD28. While in the experiments of function of Treg, we use CD4+CD25-Naive T cells as the effective cells (Tef). Group A, coculatured the Tef with PBS; Group B, coculatured the Tef with CD3/CD28; Group C, added the E2-untreated Treg to the culture with the proportion of Tef and Treg 1:1; Group D, insteaded of E2-untreated Treg with E2-treated Treg in the Group C. In vivo, we administrated pregnancy E2/P4 or saline in the ovariectomized and orchiectomized mice. After reconstituting the sex hormone to the physiological concentration range of pregnancy for two weeks, the mice were sacrificed, and lymphocytes were isolated from thymus, spleen, iliac lymph nodes and peripheral blood. The CD4+CD25+ Foxp3+ regulate T cells were detected by flow cytometry and Immunohistochemistry. Results our results revealed that E2 could significantly promote the proliferation index of labeled Treg with the costimulate signals in vitro. E2 alone could not expand the proportion of Tregs, which was not matched with the result in vivo. This phenomenon may involve in broken down of anergy of Treg by E2, and then the costimulate signals promoted the proliferation of the Tregs. In the function experiments E2 could also enhance the inhibitory effect of the effective cells. In vivo, the groups received E2 alone showed a notable increase in the proportion of the Tregs that marked CD4+CD25+ Foxp3+ in spleen, iliac lymph nodes and blood. CD4+CD25+ Foxp3+ cells constituted 10.5% of all CD4+ cells in spleen, 7.63% in iliac lymph nodes and 6.13% in blood, While the proportion in the thymus can’t be enhanced compared to the vehicle groups. The P4 administration could improve the proportion of Tregs lightly, but without statistically significant. During the group combine with E2 and P4, each tissue samples showed a same statistically significant trend in the increase of Tregs with E2 alone. Furthermore, no difference was found in the change of Tregs between the ovariectomized and orchiectomized mice. The similar results could be found in immunohistochemistry. Conclusions E2 could promote the proliferation of regulatory T cells with the TCR signals, not only expanded the amout, but also enhancede the function. In vivo, E2 can drive the expansion of the Tregs in the secondary lymphoid organs and peripheral blood, but not P4. Neither of them can improve the proportion of Tregs in thymus. No gender difference could be found in the effect, which may involve in the same distribution of hormone corelative receptors. PartⅡPaternal Antigen-Specific regulatory T cells were involved in the protection in maternal-fetal toleranceObjective We determined whether the function of T regulatorycells (Tregs) on the effector T cells was specific for paternal antigens. Methods The CBA/J×DBA/2J murine models were used as an immunological spontaneous abortion and provoked spontaneously high abortion rates.The MLC was established as follow. Effector cells from spleen of pregnant CBA/J females in abortion group were incubated with MMC treated DBA/2J males’spleen cells. Cultures were added with Tregs from normal pregnant or nonpregnant or further control (Balb/c female mated C57BL/6) mice (ratios of T effector:Treg cells were 10:1,1:1, and 1:5, respectively). Effector cells were stained with CFDA-SE before coculture,72 hours later; cells were processed and analyzed for their proliferation and cytokines. In vivo, Tregs from the normal pregnant, nonpregnant CBA/J females and further control were injected intravenously on day 0 to 2 of pregnancy to CBA/J females mated with DBA/2J abortion group. The abortion rates of each group were documented. Results A statistically significant increased percentage of Treg in peripheral blood from abortion mice when compared to non-pregnant mice (6.59% vs 3.55%, P<0.01), and also significant increased percentage of Treg in peripheral blood from normal pregnant mice when compared to abortion mice (8.34% vs 6.59%, P<0.01). The similar results could be observed in lymphoid nodes (6.86% vs 4.85; 9.62% vs 4.85%, P<0.05; 9.62% vs 6.86%, P<0.05). Cultureed with Tregs from all groups showed comparable ex vivo Treg number depended cytokine secretion such as up-regulated IL-10 and down-regulated IFN-γ.The defective function and insufficient number of Treg cells were involved in the spontaneous abortion combination of DBA/2J-mated CBA/J mice. We found that functional Treg adoptive transfer in vivo did not prevent the rejection of fetal on the abortion prone mate which was undergoing immunological abortion. Full normal pregnancy could only be achieved after adoptive transfer of Treg from normal pregnant mice, which was specific for paternal antigen. Conclusions All of the Treg cells from normal pregnant and nonpregnant CBA/J mice could inhibit the proliferation from abortion mice in vitro whereas in vivo prevention of fetal rejection could only be achieved after adoptive transfer of Treg cells from normal pregnant mice. Our data suggest that pregnancy-induced Treg cells protect the fetal from being rejected in a paternal antigen specific way. Part III Pregnancy Levels of Estrogen and Progesterone enhance regulatory responses to H-Y mismatched skin graft in murine modelObjective To investigate the effects of estrogen (E2) and progesterone (P4) alone or combined with each other to H-Y skin graft and the potential mechanism. Methods The female C57BL/6 mice were ovariectomized and divided into four groups (n=15 in each). The mice were treated consecutively for 14d with subcutaneous injection of saline, E2 and P4 alone and in combination respectively. Before and after H-Y skin grafting, half of each group was sacrificed, and the CD4+CD25+Foxp3+regulatory T cells of peripheral blood and the serum cytokines were detected by flow cytometry and ELISA, respectively. The skin grafts survivals of the other half were observed. Results The administration of sex hormone regardless of E2 and P4 alone or in combination, significantly decreased production of pro-inflammatory cytokines (IFN-y and TNF-a), but increased production of anti-inflammatory cytokines (IL-10 and TGF-β). (P<0.05) E2 alone could significantly augment the proportion of regulatory T cells. In the presence of H-Y antigen, this effect was further enhanced. (P<0.05) By contrast, P4 had no effect on the expression of Foxp3, regardless of the presence of H-Y antigen or not. (P>0.05) The skin grafts survivals were significantly prolonged in the different experimental groups compared to vehicle control group.(P<0.05) Conclusions Pregnancy Levels of E2 and P4 suppresses the inflammatory response and enhances the regulatory response to exogenous antigen, through influencing the levels of cytokines and/or the proportion of regulatory T cells. It may contribute to induce the transplant tolerance.更多还原