本文選題：支氣管哮喘 + Th2免疫應(yīng)答�。� 參考：《上海交通大學》2015年博士論文

【摘要】：背景支氣管哮喘是以Th2免疫應(yīng)答為主的慢性過敏性氣道炎癥。近年研究發(fā)現(xiàn)嗜堿性粒細胞在不同疾病模型中能夠誘發(fā)Th2免疫應(yīng)答,但其具體機制目前尚不清楚。OX40-OX40L共刺激通路在Th細胞分化及哮喘中均發(fā)揮重要作用。目的通過體內(nèi)外研究闡釋并明確嗜堿性粒細胞始動過敏性氣道炎癥的作用,在此基礎(chǔ)上進一步探討嗜堿性粒細胞啟動Th2免疫反應(yīng)誘發(fā)過敏性氣道炎癥的信號分子機制。方法1.利用OVA免疫小鼠建立哮喘氣道炎癥模型,觀察在哮喘炎癥早期小鼠縱隔淋巴結(jié)(Mediastinal lymph nodes,MLN)中嗜堿性粒細胞、樹突狀細胞(Dendritic cells,DCs)和Th2細胞的時間變化曲線和嗜堿性粒細胞表面分子表達的變化。2.建立IL-3誘導小鼠骨髓細胞定向分化為嗜堿性粒細胞的體外培養(yǎng)體系;流式細胞儀分選嗜堿性粒細胞并鑒定。在此基礎(chǔ)上觀察髓源性嗜堿性粒細胞(Bone marrow-derived basophils,BM-Bas)OX40和OX40L表達特點。3.建立體外嗜堿性粒細胞誘導Th2細胞分化體系,在此基礎(chǔ)上采用OX40L封閉性抗體(抗小鼠CD252抗體)或者OX40~-/- 小鼠na?ve T細胞,觀察在OX40-OX40L共刺激通路阻斷條件下對嗜堿性粒細胞誘導Th2細胞分化的影響。4.利用OVA致敏、激發(fā)C57BL/6小鼠構(gòu)建哮喘模型,在此基礎(chǔ)上在致敏階段尾靜脈注射OX40L阻斷性抗體(抗小鼠CD252抗體),觀察氣道病理組織學改變、血清OVA特異性Ig E(Ovalbumin-special immunoglobulin E,OVA-s Ig E)、支氣管肺泡灌洗液(Bronchial alveolar lavage fluid,BALF)中嗜酸性粒細胞數(shù)量和BALF中Th2型細胞因子IL-4、IL-5和IL-13的變化。5.建立體外過繼性回輸MLN嗜堿性粒細胞誘發(fā)小鼠哮喘模型,觀察阻斷OX40-OX40L共刺激通路后受體小鼠MLN Th2細胞比例變化及氣道病理組織學、血清OVA-s Ig E、BALF中嗜酸性粒細胞數(shù)量和Th2型細胞因子IL-4、IL-5和IL-13的變化。結(jié)果1.在哮喘炎癥早期,MLN中嗜堿性粒細胞數(shù)目顯著增加,而DCs無明顯變化,并且嗜堿性粒細胞數(shù)達到峰值時間早于Th2細胞。同時嗜堿性粒細胞表面OX40L信號分子變化最為顯著。2.體外IL-3誘導BM-Bas并受DNP-OVA和抗DNP-Ig E復合物刺激后誘導性表達OX40L。3.體外阻斷OX40-OX40L共刺激通路減少嗜堿性粒細胞誘導Th2細胞分化比例。4.致敏階段抗小鼠CD252抗體阻斷OX40-OX40L共刺激通路降低哮喘小鼠血清OVA-s Ig E、減少BALF中嗜酸性粒細胞數(shù)量、降低BALF中IL-4、IL5和IL-13水平,減輕肺組織過敏性氣道炎癥。5.體外過繼性回輸MLN嗜堿性粒細胞啟動Th2免疫應(yīng)答誘發(fā)小鼠哮喘炎癥,而OX40~-/- 受體小鼠和回輸OX40L處理的嗜堿性粒細胞的WT C57BL/6受體小鼠MLN中Th2細胞比例降低,肺部過敏性氣道炎癥減輕。結(jié)論1.嗜堿性粒細胞早期參與始動過敏性氣道炎癥。2.嗜堿性粒細胞在體內(nèi)外誘導性表達OX40L。3.OX40-OX40L共刺激通路介導嗜堿性粒細胞誘導Th2細胞分化。4.在OVA誘導的過敏性氣道炎癥中,OX40-OX40L共刺激通路介導嗜堿性粒細胞誘發(fā)Th2免疫應(yīng)答,導致過敏性氣道炎癥。
[Abstract]:Background bronchial asthma is a chronic allergic airway inflammation based on Th2 immune response. In recent years, it has been found that basophil can induce Th2 immune response in different disease models, but its specific mechanism is not clear. OX40-OX40L costimulatory pathway plays an important role in Th cell differentiation and asthma. Objective to elucidate and clarify the role of eosinophil in allergic airway inflammation in vitro and in vivo, and to further explore the signaling molecular mechanism of allergic airway inflammation induced by basophil initiating Th2 immune reaction. Method 1. The asthma airway inflammation model was established in mice immunized with OVA. The basophil in mediastinal lymph nodesMLNs was observed in the mediastinal lymph in the early stage of asthmatic inflammation in mice. Dendritic cells (Th2) and the changes of basophilic granulocyte surface molecular expression. 2. An in vitro culture system of IL-3 induced bone marrow cells to differentiate into basophil was established, and basophil was separated and identified by flow cytometry. On this basis, the expression characteristics of bone marrow-derived basophils BM-Baso OX40 and OX40L in medullary basophilic granulocytes were observed. The differentiation system of Th2 cells induced by basophilic granulocytes in vitro was established. On this basis, OX40L closed antibody (anti-mouse CD252 antibody) or OX40- / -r-mouse na?ve T cell were used. To observe the effect of blocking OX40-OX40L costimulatory pathway on the differentiation of Th2 cells induced by basophil. 4. 4. The asthmatic model of C57BL/6 mice was established by sensitizing with OVA, and the airway histopathological changes were observed by injecting OX40L blocking antibody (anti-mouse CD252 antibody) into caudal vein at sensitizing stage. The changes of eosinophils and Th2 type cytokines IL-4IL-5 and IL-13 in serum OVA specific Ig E(Ovalbumin-special immunoglobulin and bronchoalveolar lavage fluid (BALF) and Th2 type cytokines (IL-4, IL-5 and IL-13) in bronchoalveolar lavage fluid (bronchoalveolar lavage fluid, bronchoalveolar lavage fluid) and Th2 type cytokines (IL-4, IL-4, IL-5 and IL-13) were observed. To establish a mouse asthma model induced by adoptive reinjection of MLN basophils in vitro and observe the changes of MLN Th2 cell ratio and airway histopathology after blocking OX40-OX40L co-stimulation pathway in mice. The changes of eosinophil count and Th2 type cytokine IL-4 IL-5 and IL-13 in serum OVA-s Ig euclif. Result 1. In the early stage of asthma inflammation, the number of basophilic granulocytes in MLN increased significantly, but the number of DCs did not change, and the peak time of the number of basophil was earlier than that of Th2 cells. At the same time, the change of OX40L signal molecules on basophil surface was the most significant. 2. 2. BM-Bas was induced by IL-3 in vitro and stimulated by DNP-OVA and anti DNP-Ig E complex. Blocking OX40-OX40L costimulatory pathway in vitro reduced the percentage of differentiation of Th2 cells induced by basophil. 4. 4. The blocking of OX40-OX40L costimulatory pathway by CD252 antibody in sensitized mice decreased serum OVA-s Ig E, decreased the number of eosinophils in BALF, decreased the levels of IL-4 IL-5 and IL-13 in BALF, and alleviated allergic airway inflammation of lung tissue. MLN basophilic granulocytes initiated Th2 immune response to induce asthma inflammation in mice in vitro, while the percentage of Th2 cells in MLN of OX40 + -r-receptor mice and WT C57BL/6 receptor mice treated with OX40L was decreased. Allergic airway inflammation in the lungs is alleviated. Conclusion 1. Basophilic granulocytes are involved in allergic airway inflammation. 2. 2. In vitro and in vivo expression of OX40L.3.OX40-OX40L costimulatory pathway mediated differentiation of Th2 cells induced by basophil. In allergic airway inflammation induced by OVA, OX40-OX40L costimulatory pathway mediates Th2 immune response induced by basophil, leading to allergic airway inflammation.
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R725.6

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