OX40-OX40L共刺激通路介导嗜碱性粒细胞始动过敏性气道炎症的研究

发布时间：2018-05-29 16:41

本文选题：支气管哮喘 + Th2免疫应答　；参考：《上海交通大学》2015年博士论文

【摘要】：背景支气管哮喘是以Th2免疫应答为主的慢性过敏性气道炎症。近年研究发现嗜碱性粒细胞在不同疾病模型中能够诱发Th2免疫应答,但其具体机制目前尚不清楚。OX40-OX40L共刺激通路在Th细胞分化及哮喘中均发挥重要作用。目的通过体内外研究阐释并明确嗜碱性粒细胞始动过敏性气道炎症的作用,在此基础上进一步探讨嗜碱性粒细胞启动Th2免疫反应诱发过敏性气道炎症的信号分子机制。方法1.利用OVA免疫小鼠建立哮喘气道炎症模型,观察在哮喘炎症早期小鼠纵隔淋巴结(Mediastinal lymph nodes,MLN)中嗜碱性粒细胞、树突状细胞(Dendritic cells,DCs)和Th2细胞的时间变化曲线和嗜碱性粒细胞表面分子表达的变化。2.建立IL-3诱导小鼠骨髓细胞定向分化为嗜碱性粒细胞的体外培养体系;流式细胞仪分选嗜碱性粒细胞并鉴定。在此基础上观察髓源性嗜碱性粒细胞(Bone marrow-derived basophils,BM-Bas)OX40和OX40L表达特点。3.建立体外嗜碱性粒细胞诱导Th2细胞分化体系,在此基础上采用OX40L封闭性抗体(抗小鼠CD252抗体)或者OX40~-/- 小鼠na?ve T细胞,观察在OX40-OX40L共刺激通路阻断条件下对嗜碱性粒细胞诱导Th2细胞分化的影响。4.利用OVA致敏、激发C57BL/6小鼠构建哮喘模型,在此基础上在致敏阶段尾静脉注射OX40L阻断性抗体(抗小鼠CD252抗体),观察气道病理组织学改变、血清OVA特异性Ig E(Ovalbumin-special immunoglobulin E,OVA-s Ig E)、支气管肺泡灌洗液(Bronchial alveolar lavage fluid,BALF)中嗜酸性粒细胞数量和BALF中Th2型细胞因子IL-4、IL-5和IL-13的变化。5.建立体外过继性回输MLN嗜碱性粒细胞诱发小鼠哮喘模型,观察阻断OX40-OX40L共刺激通路后受体小鼠MLN Th2细胞比例变化及气道病理组织学、血清OVA-s Ig E、BALF中嗜酸性粒细胞数量和Th2型细胞因子IL-4、IL-5和IL-13的变化。结果1.在哮喘炎症早期,MLN中嗜碱性粒细胞数目显著增加,而DCs无明显变化,并且嗜碱性粒细胞数达到峰值时间早于Th2细胞。同时嗜碱性粒细胞表面OX40L信号分子变化最为显著。2.体外IL-3诱导BM-Bas并受DNP-OVA和抗DNP-Ig E复合物刺激后诱导性表达OX40L。3.体外阻断OX40-OX40L共刺激通路减少嗜碱性粒细胞诱导Th2细胞分化比例。4.致敏阶段抗小鼠CD252抗体阻断OX40-OX40L共刺激通路降低哮喘小鼠血清OVA-s Ig E、减少BALF中嗜酸性粒细胞数量、降低BALF中IL-4、IL5和IL-13水平,减轻肺组织过敏性气道炎症。5.体外过继性回输MLN嗜碱性粒细胞启动Th2免疫应答诱发小鼠哮喘炎症,而OX40~-/- 受体小鼠和回输OX40L处理的嗜碱性粒细胞的WT C57BL/6受体小鼠MLN中Th2细胞比例降低,肺部过敏性气道炎症减轻。结论1.嗜碱性粒细胞早期参与始动过敏性气道炎症。2.嗜碱性粒细胞在体内外诱导性表达OX40L。3.OX40-OX40L共刺激通路介导嗜碱性粒细胞诱导Th2细胞分化。4.在OVA诱导的过敏性气道炎症中,OX40-OX40L共刺激通路介导嗜碱性粒细胞诱发Th2免疫应答,导致过敏性气道炎症。
[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.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R725.6

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