本文關鍵詞： 雌激素 孕激素 調節(jié)性T細胞 性別 小鼠 母胎耐受 調節(jié)性T細胞 妊娠 抗原特異性 小鼠 雌激素 孕激素 調節(jié)性T細胞 H-Y抗原 小鼠　出處：《華中科技大學》2010年博士論文　論文類型：學位論文

【摘要】： 第一部分妊娠水平的雌、孕激素對機體免疫系統(tǒng)的影響及機制研究 目的探討妊娠濃度雌、孕激素對小鼠體內調節(jié)性T細胞的影響及可能機制。 方法實驗分為體內部分和體外部分。體內實驗：建立去勢C57BL／6小鼠模型,兩周后分別予以皮下注射雌激素(E2)、孕激素(P4)、雌孕激素聯(lián)合及溶劑(生理鹽水)模擬重建妊娠濃度小鼠激素水平。維持兩周后,流式細胞術和免疫組化檢測給藥后小鼠外周血、脾臟、腹腔淋巴結、胸腺中調節(jié)性T細胞(Treg)比例變化情況。并通過ELISA的方法檢測外周血中TNF-α、IL-2、IFN-γ、IL-10、TGF-β等細胞因子水平的變化。體外實驗：通過CFSE標記磁珠分選的調節(jié)性T細胞反應細胞,外加E2和共刺激信號,觀察標記的調節(jié)性T細胞是否發(fā)生增殖,并進一步通過CFSE標記法結合單向混合淋巴細胞培養(yǎng)來驗證其調節(jié)性T細胞的功能。 結果體內實驗：無論雌、雄小鼠,去勢后單獨的妊娠水平的E2即可明顯提高外周血、脾臟、腹腔淋巴結中CD4+CD25+Foxp3+Treg占CD4+T細胞的比例(分別為6.13±1.32%；10.50±1.85%；7.63±1.68%),與溶劑組相比有明顯統(tǒng)計學差異(P0.05)。但在胸腺中CD4+CD25+Foxp3+Treg的比例與溶劑組沒有統(tǒng)計學差異。與此同時,單獨的妊娠水平的P4相比于溶劑組,Treg比例有所升高,但不具有統(tǒng)計學意義(P0.05)。而聯(lián)合應用雌、孕激素對Treg的影響與單獨應用E2的結果類似,兩者沒有統(tǒng)計學差異(P0.05)。相同干預藥物,Treg的比例變化在雌雄兩組之間沒有差異(P0.05)。同樣的結果在免疫組化中得到驗證。對于外周血中細胞因子的影響,在沒有外來抗原刺激的條件下是沒有多大影響的。 體外實驗：單獨的E2處理對于調節(jié)性T細胞并不能直接誘導CFSE標記的調節(jié)性T細胞的增殖(PI=1.01±0.01),單獨的TCR雙信號刺激也不能檢測到CFSE的增殖(PI=1.03±0.02),只有在E2的預處理聯(lián)合CD3/CD28的刺激下才能檢測到調節(jié)性T細胞自身的增殖(PI=1.15±0.03)。驗證了E2對于調節(jié)性T細胞數(shù)量上的促增殖作用之后,功能學方面的實驗結果提示,CD3／CD28的刺激能顯著促進CD4+CD25- NaiveT細胞的增殖(PI=1.56±0.03),在此基礎上按照反應細胞與調節(jié)性T細胞比為1：1,未經E2處理組也能檢測到對刺激后增殖的抑制(PI=1.18±0.04),E2的預處理能進一步增強調節(jié)性T細胞的抑制功能(PI=1.07±0.02),不僅與刺激組相比有差異(1.07±0.02 vs 1.56±0.03,P0.05),與未經E2處理組同樣有差異(1.07±0.02 vs 1.18±0.04,P0.05)證明了E2不僅能通過促進調節(jié)性T細胞的增殖而擴增,而且在功能上也能進一步增強其對效應T細胞活化后增殖的抑制效應。 結論單獨應用妊娠濃度E2可明顯提高小鼠外周血,脾臟,腹腔淋巴結CD4+CD25+Foxp3+Treg比例,妊娠濃度的P4對外周Treg沒有影響，，兩種激素對中樞淋巴器官的淋巴細胞均沒有影響。這可能與小鼠T淋巴細胞中雌、孕激素的受體分布、功能及與性別遺傳沒有相關性有關。體外實驗的結果不僅驗證妊娠水平的E2通過改變調節(jié)性T細胞的無反應性,在聯(lián)合TCR信號下能促進調節(jié)性T細胞自身發(fā)生增殖,還能在功能上加強調節(jié)性T細胞的抑制作用。無論是中樞還是外周的調節(jié)性T細胞的比例均沒有影響.妊娠水平的雌、孕激素對于血清中細胞因子免疫網(wǎng)絡是沒有太大的影響。 第二部分調節(jié)性T細胞在妊娠保護中的作用及抗原特異性的研究 目的：探討妊娠中調節(jié)性T細胞的免疫調節(jié)作用是否具有抗原特異性。 方法：本實驗分體外部分和體內部分。體外實驗：以雌性CBA/J與雄性DBA/2J同籠構建流產趨勢模型,從流產趨勢孕10天雌鼠脾臟分離的單個核細胞經尼龍毛處理后作為效應T細胞(Tef)；DBA雄鼠脾細胞經MMC處理后作為APC；根據(jù)培養(yǎng)體系中調節(jié)性T細胞(Treg)的來源分組：未孕CBA雌鼠來源的Treg為未孕組,正常妊娠(CBA/J×Balb/c)來源的Treg為正常妊娠組,無關正常妊娠(Balb/c×C57BL/6)來源的Treg為正常妊娠無關對照組。按照Tef與Treg比分別為10：1,1：1,1：5構建單向混合淋巴細胞培養(yǎng)體系,72小時后,流式細胞儀檢測標記細胞的增殖指數(shù)(Proliferative Index, PI), ELISA檢測細胞因子水平。 體內實驗：構建流產趨勢的妊娠模型,于妊娠第0-2天,過繼輸注來自正常妊娠組、未孕組、無關妊娠對照組的CD4+CD25+Treg,觀察Treg的過繼輸注對妊娠結局的影響。按照Treg的來源不同分為：正常妊娠組、未孕組、無關妊娠對照組。 結果：體外實驗：在Tef與Treg比例為10：1的體系中,未孕組效應性T細胞增殖明顯受到抑制。在1：1,1：5的體系中同樣均能檢測到明顯的抑制效應,但這種抑制效應沒有劑量依賴性。而分別來自于其他兩組正常妊娠小鼠的Treg也表現(xiàn)出相似的增殖抑制效應,各組之間沒有統(tǒng)計學差異。在Treg的干預后培養(yǎng)體系中細胞因子水平的變化在Treg的干預后,IL-10的水平明顯升高,并且這種水平的升高呈Treg劑量依賴性正相關。而IFN-γ的水平明顯減少,并且與Treg劑量呈負相關。 體內實驗：在外周血中,流產趨勢組CD4+CD25+Foxp3+Treg細胞占CD4+T細胞的比例較未孕組明顯增高(6.59% vs 3.55%,P0.05)；正常妊娠組中外周血能檢測到更為顯著的Treg的升高,不僅與未孕組有差異(8.34% vs 3.55%,P0.05),與流產趨勢組亦有明顯差異(8.34% vs 6.59%,P0.05)。同樣,在淋巴結(6.86% vs 4.85；9.62%vs 4.85%, P0.05; 9.62% vs 6.86%, P0.05)中也獲得相似結果。過繼輸注后,只有來自正常妊娠組小鼠的Treg的過繼輸注才能完全阻止流產的發(fā)生。而來自未孕組和無關對照組的Treg不能逆轉流產的發(fā)生。Treg過繼輸注不能改變體內Th1型細胞因子水平,但是能顯著提升IL-10水平。 結論：無論哪種來源的Treg都具有免疫調節(jié)的潛能。在體外均能檢測到對Treg增殖的抑制效應,但并沒有表現(xiàn)出抗原特異性的更為顯著抑制功能；Treg在體外可能是通過分泌高濃度的IL-10發(fā)揮抑制效應。但在體內正常的妊娠過程中產生的Treg在保護胚胎免受母體攻擊方面是具有抗原特異性的,只有之前接觸胎兒父系抗原的具有抗原特異性的Treg才能在體內發(fā)揮對胎兒的保護作用。 第三部分妊娠水平的雌、孕激素在H-Y皮膚移植中的作用及其機制研究 目的探討妊娠濃度雌激素(E2)、孕激素(P4)對小鼠H-Y皮膚移植的影響及其機制。 方法建立小鼠去勢模型,每日分別注射E2、P4及E2+P4聯(lián)合注射,14d后受鼠行H-Y皮膚移植并繼續(xù)給藥。檢測移植前及移植后第3天外周血中CD4+CD25+Foxp3+調節(jié)性T細胞(Treg)變化及外周血細胞因子水平；觀察H-Y皮膚移植物的存活。 結果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的比例進一步升高(11.78±2.32% vs 3.33±0.57%,P0.05)；P4對Treg沒有顯著影響(4.01±0.79% vs 3.26±0.68%,P0.05),而移植前后E2+P4聯(lián)合對Treg的影響與單獨應用E2結果相似(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對Treg仍無明顯影響,而E2+P4聯(lián)合亦不能進一步提高Treg的比例。細胞因子檢測顯示,E2、P4均能減少促炎因子分泌,并提高抗炎因子分泌(P0.05)。E2、P4均能有效延長H-Y皮膚移植物存活((44.0±1.2天,35.5±0.8天,23.0±1.6天,P0.05,P0.05),且兩者聯(lián)用具有協(xié)同效應(MST=56.5±3.2天)。結論P4可通過誘導細胞因子免疫偏移促進H-Y皮膚移植物的存活。而E2除誘導細胞因子免疫偏移外,還通過提高Treg水平而延長移植物的存活。
[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.
緇撴灉E2鍙彁楂樺鍛ㄨTreg鐨勬瘮渚

本文編號：1504934