TIM蛋白论文：TIM蛋白 树突状细胞 Th2细胞 食物过敏 金黄色葡萄球菌肠毒素B 卵清蛋白

TIM蛋白论文：TIM蛋白 树突状细胞 Th2细胞 食物过敏 金黄色葡萄球菌肠毒素B 卵清蛋白 TIM蛋白论文:TIM蛋白 树突状细胞 Th2细胞 食物过敏 金黄色葡萄球菌肠毒素B 卵清蛋白 【中文摘要】随着新型抗生素和疫苗的发展及其在临床上的应用,感染性疾病的治疗已取得巨大成功,但在过去的几十年,过敏性疾病却呈现出明显上升趋势,大约15%-20%的人对外源抗原产生1gE介导的过敏反应,其中约有2%-6%的人存在食物过敏(food allergy, FA) 及相关症状。近年虽然过敏性疾病成为研究热点且已有较多的研究,但是过敏性疾病的发病机制及病因学仍不清楚,其治疗方法有限且效果不佳。研究显示树突状细胞(dendritic cell, DC)和Th2细胞在过敏免疫反应中具有关键作用。DC是机体内功能最强的抗原提呈细胞(antigen presenting cell, APC),也是唯一能激活初始T细胞的APC, DC捕获和加工外源性抗原,并递呈抗原信息给T细胞,激活T细胞介导的免疫反应。成熟的DC高表达MHC-?/?类分子、共刺激分子如CD80和CD86等协助抗原信息的递呈,有效活化T细胞。初始CD4+T细胞被激活后分化为Th1、Th2. Th17或Treg细胞,Th2细胞主要释放细胞因子IL-4、IL-5和IL-13。这些细胞因子不仅在诱导B细胞产生抗原特异性IgE中起重要作用,而且在粘液分泌、肌肉收缩、和嗜酸性粒细胞增多中起重要作用。当再次暴露于特异性抗原后,IgE交叉连接激活肥大细胞释放过敏性介质,促发过敏性反应和产生过敏性临床症状。因此Th2细胞的极化是食物过敏免疫机制中重要一步。T细胞免疫球蛋白与黏蛋白域蛋白(T cell immunoglobulin and mucin domains, TIMs)基因家族是一组新的细胞表面蛋白家族。已发现TIMs基因家族在小鼠中包括8个成员(TIMs 1-8)和在人类中3个成员(TIM1,3和4)。研究显示TIMs在过敏性疾病和自身免疫性疾病中起重要的调节作用。其中TIM4表达于APC,尤其是成熟的DC, TIM1表达于T细胞,特别是Th2细胞,近来发现它们是T细胞重要的调节因子。体外研究显示DC中TIM4蛋白的表达与自身免疫性疾病的严重程度正相关,TIM4结合TIM1共同促进T细胞的增殖,且提示TIM4可能作为一个新的分子来调节T细胞反应。我们前期研究,显示在过敏小鼠中存在Treg细胞功能不全,TIM4与TIM1的相互作用可降低Treg细胞的功能及状态,破坏免疫耐受平衡。但是TIM4对食物抗原特异性Th2细胞分化的影响研究甚少,体外实验证据不多,其机制仍需进一步研究。我们推测微生物产物和食物抗原共同作用能够导致FA,其中TIM4与其受体的相互作用可能导致Th1/Th2细胞失衡和免疫耐受的打破,是引起FA的关键。本研究将通过体外培养BMDC,与CD4+T细胞共同培养来研究微生物产物和食物抗原对CD4+T细胞的影响,并应用TIM4抗体干预,探讨TIM4在FA发病过程中的作用,从而进一步了解食物过敏发生的分子机制,并为临床治疗提供理论依据。通过TIM4 mRNA的表达,DC表面分子CD11c、MHC-?、CD86的表达,研究微生物产物对TIM4的作用和微生物产物对DC的刺激作用。使用SEB和OVA共同作用于BALB/c小鼠,分离过敏小鼠脾脏CD4+T细胞,和DC在不同条件下共同培养,检测细胞上清液中Th1/Th2细胞因子的水平。探讨在微生物产物参与的食物过敏(food allergy, FA)中TIM4对 CD4+T细胞分化的影响。方法培养BALB/c小鼠骨髓来源的树突状细胞(bone marrow-derived DC, BMDC),培养的DC分为两组:金黄色葡萄球菌肠毒素B(SEB)刺激组和空白对照组。使用SEB和OVA致敏BALB/c小鼠,取过敏模型小鼠脾脏CD4+T细胞,和DC细胞共同培养分为5组:空白对照组、SEB+卵清蛋白(OVA)共同作用组、SEB组、OVA组及TIM4抗体干预组。进行相关指标的检测。1. RT-PCR检测DC的TIM4 mRNA表达。2.流式细胞仪检测DC表面CDIIcn MHC-?、CD86的表达。3. ELISA法测定混合培养细胞上清液IL-4与IFN-y的表达。所得数据均采用SPSS13.0统计软件包进行。组间均值比较采用单因素方差分析,以a=0.05为假设检验。结果1.体外分离骨髓细胞,诱导分化为树突状细胞,通过流式细胞仪检测特征性标志CDllc纯度可达70%,并具有特征性形态。2.使用SEB刺激DC,与空白对照组相比,SEB刺激组DC TIM4 mRNA表达明显增高(0.941?0.018 vs 0.422?0.083,均P<0.05),并具有剂量依从性。3.使用SEB刺激DC,流式细胞仪检测:SEB刺激组DC表面分子MHC-?、CD86表达明显高于空白对照组(MHC-?:76.684%?3.1803% vs 52.984%?3.6026%,P=0.000;CD86:89.746%?2.113%vs 67.558%?0.4341%,P=0.000)。4.DC和CD4+T细胞共同培养中,相比对照组,SEB+OVA组IL-4表达显著增高(295.834?20.408 vs 78.335?13.109,均P<0.05),而IFN-γ的表达明显减少(362.109?92.271 vs 761.897?102.967,均P<0.05);SEB组或OVA组IL-4和IFN-γ与空白对照组无明显差异;DC和CD4+T细胞共同培养中应用TIM4抗体干预后,结果显示对比SEB+OVA组IL-4 明显降低,而IFN-γ的表达明显增高(P均<0.05),与对照组、SEB组 和OVA组相比两种细胞因子的表达无统计学差异。结论1.SEB刺激 DC增加TIM4的表达。2.SEB刺激DC促进DC上MHC-?和共刺激分子 CD86表达上调,促进DC功能成熟。3.SEB和OVA共同刺激促进CD4+T 细胞分化为Th2细胞,共同导致食物过敏。单一的SEB或者OVA不能 引起食物过敏反应。4.TIM4抗体干预抑制Th2细胞分化,有效纠正 Th1/Th2细胞失衡,治疗食物过敏,显示TIM4在食物过敏反应中起重 要作用。 【英文摘要】BACKGROUNDWith the development and clinical application of new types of antibiotic and vaccine, the treatment of infectious diseases have achieved a great success. However, in the past a few decades, the prevalence of allergic diseases have grown obviously, as much as 15%to 20%of the population have IgE-mediated allergic reactions with exogenous antigens, and as much as 2%to 6%of the population have food allergy and allergic related symptoms. In recent years, although allergic diseases have been studied numerously, the pathogenesis and etiology of them remain unclear and the treatment of them are limited and ineffective.It is generally believed that dendritic cells and Th2 cells are critical in the allergic immune response. DCs are the most powerful antigen presenting cells (APCs) in the body, which are the only APCs that can activate the naive T cells. DCs capture and process the exogenous antigens, and present antigen information to T cells to initialize T cell-mediated immune response. The mature DCs can express high levels of MHC-?/?molecules and costimulatory molecules such as CD80 and CD86 which can assist DCs present antigen to T cells, and then activate T cells effectively. The naive CD4+T cells could be activated to differentiate into Thl, Th2, Th17 or Treg cells. Thl cells mainly release type 1 cytokines, such as IFN-γ. While Th2 cells mainly release IL-4, IL-5 and IL-13. Then These cytokines play an important role not only in the production of antigen-specific IgE by B cells, but also in mucus secretion, muscle contractility, and eosinophilia. Mast cells activated by IgE cross-linking release allergic mediators to initiate the hypersensitivity reactions and allergic clinical symptoms on re-exposure to specific antigens. Thus, the activation of Th2 cells is a critical step in the immune mechanisms of food allergy.Recent studies discovered that T cell immunoglobulin and mucin domains (TIMs) gene family introduced a new family of cell surface proteins. The TIMs gene family have been found including 8 members (TIMs 1-8) in mice, and including three members (TIM1,3 and 4) in humans. They have important regulatory role in the development of allergic diseases and autoimmune diseases. TIM4 is expressed on the surface of APC, especially mature DCs, and TIM1 is expressed on T cells, particularly Th2 cells. It is recently found that they are the important factors of T cells. In vitro studies have shown that the expression of TIM4 protein on DCs is related to the severity level of autoimmune diseases. TIM4 that combines with TIM1 promotes T cell proliferation, and studies suggest that TIM4 may as a new molecular to regulate T cell response. Our previous studies showed that food allergic mice existed Treg cell dysfunction, the interaction of TIM4 and TIM1 reduce the function and status of Treg cell and break the balance of intestinal immune tolerance. But whether TIM4 effect the differentiation of food antigen-specific Th2 cells keep poorly studied, more experimental evidences in vitro should be provided, and the underlying mechanism needs further study.It is supposed that concurrent exposure to both microbial product and food antigen leads to the development of food antigen-specific Th2 cells and food allergy (FA). the interaction of TIM4 and its receptor may result in the imbalance of Thl/Th2 and break immune tolerance, which is the key pathogenesis of FA. In this study, BMDC and CD4+T cells were cocultured, evaluate effective of microbial product and food antigen for CD4+T cells and the role of TIM4 in the FA. Through this study, the molecular mechanism of food allergy could be futher understood and the new theoretical basis may be provided for clinical treatment of the FA. AIMSThe expression of TIM4 mRNA was measured and the expressions of CD llc, MHC-?, CD86 on DCs were detected to investigate the role of microbial product in the generation of TIM4 and the stimulant effect of microbial product on DCs. The splenic CD4+T cells were isolated from the allergic BALB/c mice that were sensitized together by SEB and OVA, and CD4+T cells and DCs were cultured under different conditions in vitro in order to test the cytokine levels of Thl/Th2. The role of TIM4 in CD4+T reaction and food allergy was examined by exposure to microbial product.METHODSThe bone marrow-derived DCs in BALB/C mice were isolated and cultured in vitro, cultured DCs were divided into staphylococcal enterotoxin B (SEB)-stimulated group and control group. BALB/c mice were sensitized to SEB and OVA for the allergic spleen CD4+T cells. CD4+T cells and DCs were divided into 5 groups:control group, SEB plus ovalbumin (OVA) group, SEB group, OVA group and anti-TIM4 antibody plus SEB and OVA group. The related indicators were detected.1. The expression of TIM4 mRNA on DCs was detected by RT-PCR.2. The expressions of the CDllc, MHC-?, CD86 on DCs surface were analyzed by Flow cytometry.3. The levels of IL-4 and IFN-γ in mixed cultured cells supernatant were tested by ELISA.Data were expressed as the means?D. All statistical analysis was performed using SPSS statistical version 13.0. Differences between groups were determined with one-way ANOVA; a P value of less than 0.05 was considered to be statistically significant.RESULTS1. Bone marrow cells in vitro were isolated, and were induced into dendritic cells. The characteristic sign of CD llc was measured by flow cytometry, and its purity was up to 70%. DCs showed a characteristic morphology. 2. The SEB was used to stimulate DCs. The expression of TIM4 in DCs at messenger RNA (mRNA) was increased significantly in the SEB-stimulated group compared with the control group (0.941?0.018 vs 0.422?0.083, P<0.05), and the SEB up-regulated the expression of TIM-4 in a dose-dependent manner.3. The SEB was used to stimulate DCs, and the flow cytometry was used to measure. The expression of MHC-?and costimulatory molecule CD86 on DCs significantly increased after SEB stimulation (MHC-?:76.684%?3.1803%vs 52.984%?3.6026%, P= 0.000; CD86:89.746%?2.113%vs 67.558%?0.4341%, P= 0.000).4. Compared with control DCs co-culture with CD4+T cells, the level of IL-4 in culture medium in the SEB plus OVA group increased significantly (295.834?20.408 vs 78.335?13.109, P<0.05), level of IFN-y decreased significantly (362.109? 92.271 vs 761.897?102.967, P 0.05). Levels of IL-4 and IFN-y in the SEB group and OVA group showed no significant differences with those in the control group. In contrast, the level of IL-4 was significantly lower and and that of IFN-y was significantly higher in the anti-TIM4 antibody group compared with that of SEB plus OVA group (P,0.05). The expressions of IL-4 and IFN-y in the anti-TIM4 antibody group showed no significant differences with those in the control group, SEB group and OVA group.CONCLUSIONS1. SEB up-regulates the expression of TIM4 on DCs.2. SEB increases the expressions of MHC-?and CD86 on DCs, and enhances DCs maturation.3. Concurrent exposure to SEB and OVA in coculture of CD4+T cells and DCs are able to cause Th2 cells polarization and decreased Thl cytokine response, and trigger the subsequent development of food allergy. SEB or OVA alone does not change the levels of IL-4 and IFN-y, which suggest that SEB or OVA alone does not cause food allergic immune response.4. TIM4 antibody can inhibit Th2 cells polarization, it can effectively restore the imbalance of Thl/Th2 cells and it may cure food allergy, which also suggest that TIM4 is an important molecular in the pathogenesis of FA. 【关键词】TIM蛋白 树突状细胞 Th2细胞 食物过敏 金黄色葡 萄球菌肠毒素B 卵清蛋白 【英文关键词】TIM protein dendritic cell Th2 cell food allergy staphylococcal enterotoxin B ovalbumin 【目录】TIM4对小鼠食物过敏模型中抗原特异性Th2细胞分化 的影响 摘要 5-9 Abstract 9-12 前言 15-18 与方法 18-28 结果 28-31 讨论 31-38 结论 38-39 附图 39-41 参考文献 41-45 树突状细胞 和T细胞免疫应答 45-66 参考文献 58-66 中英文缩略 词对照表 66-67 在学期间发表的学术论文 67-68 致谢 68 【索购全文找】1.3.9.9.3.8.8.4.8 1.3.8.1.1.3.7.2.1 同时提供论文写作一对一辅导和论文发表服务。 【说明】本文仅为中国学术文献总库合作提供，无涉版权。作者如有异议请与总库或学校联 系。