尘螨过敏原Derf31诱发过敏性哮喘的机制研究

发布时间：2018-06-13 16:14

本文选题：Derf31 + 过敏性哮喘　；参考：《深圳大学》2017年硕士论文

【摘要】：背景:近来研究表明,过敏性哮喘的发生与遗传、生活方式及所处环境相关。在众多因素中,吸入性过敏原是主要的诱因,而且具有区域差异性。研究发现,在我国尘螨(Dust mite)是诱发过敏性哮喘的主要因素,如粉尘螨(Dermatophagoides farina)其分泌物、尸体分解物和排泄物等都能诱发过敏性疾病(荨麻疹、过敏性鼻炎、哮喘、皮炎等)。有60%成年人哮喘患者对尘螨过敏,在儿童中比例更高,约80%的儿童哮喘患者对尘螨过敏。当过敏原通过呼吸道进入肺部后,肺上皮细胞产生胸腺基质淋巴细胞生成素(TSLP)、IL-33,它们可以上调二型固有淋巴细胞(ILC2s),研究发现ILC2s与过敏性疾病有着密切的关系。目的:在前期研究中,已成功鉴定出Derf31是粉尘螨新过敏原。本研究通过纯化柱富集Derf31蛋白,从细胞实验和动物实验两部分探讨Derf31诱发过敏性哮喘的机制。方法:(1)利用原核表达系统经Ni柱纯化后获得较为纯净的Derf31和Der f 1蛋白。(2)细胞实验:Derf31与DC2.4孵育流式检测表面分子表达情况;Derf31与BMDC、脾细胞共孵育检测IL-4、IFN-γ分泌水平,CFSE法检测细胞增殖情况;TSLP通路检测:Derf31分别与正常小鼠肺组织细胞、A549共孵育,分别加入TLR2抗体、TLR4抑制剂刺激后检测IL-33、TSLP表达水平。(3)动物实验:利用Derf31、Der f 1过敏原分别构建过敏性哮喘模型,通过血清特异性抗体、细胞因子、肺部病理切片染色等验证模型是否成功;通过ELISA法检测肺部灌洗液、组织匀浆上清中的IL-33和TSLP含量,Siglec-F、CD11c流式抗体标记肺泡灌洗液中嗜酸性粒细胞,流式细胞术检测其含量;用PE-CD90、PerCP-IL-33R、FITC-Lineage和APC-CD45抗体标记并检测肺部组织ILC2细胞,同时抽取肺组织总RNA,RT-PCR检测IL-33、TSLP和IL-13的含量。结果:通过原核表达系统成功获得较高浓度的Der f 1和Derf31蛋白。Derf31能够增强DC细胞表面分子CD40、CD83和CD80的表达,同时促进Th2细胞的极化与增殖。在Derf31与肺上皮细胞共孵育体系中加入TLR2抗体后,TSLP、IL-33水平明显下降,但加入TLR4抑制剂并没有影响。Derf31可成功诱发小鼠过敏性哮喘,并且Derf31组与Der f 1组比较,其嗜酸性粒细胞、IL-33和TSLP水平显著升高;同时与空白组对比ILC2细胞表达明显增加。结论:通过本研究发现,Derf31可上调DC细胞表面的共刺激因子并促使Th0向Th2偏移;Derf31可通过TLR2调节肺部上皮细胞TSLP、IL-33的表达;在动物实验中,Derf31可诱导明显的嗜酸性粒细胞型过敏性哮喘,而且使ILC2细胞表达增加。
[Abstract]:Background: recent studies have shown that the occurrence of allergic asthma is related to heredity, lifestyle and environment. In many factors, inhalation allergens are the main inducements and regional differences. The study found that in China, Dust mite is the main factor inducing allergic asthma, such as dust mites (Dermatophagoides farina). Secretions, decomposable corpses and excreta can all induce anaphylaxis (urticaria, allergic rhinitis, asthma, dermatitis, etc.). 60% adult asthmatics are allergic to dust mites, higher in children, and about 80% of children allergic to dust mites. When the allergen passes through the respiratory tract, the lung epithelial cells produce thymic matrix Lymphocytoin (TSLP), IL-33, which can up-regulation two type of innate lymphocyte (ILC2s). Studies have found that ILC2s has a close relationship with allergic diseases. Objective: in the previous study, it has been successfully identified that Derf31 is a new allergens of the dust mites. This study has enriched Derf31 protein from the purified column, from the cell experiment and the animal experiment two parts. The mechanism of Derf31 induced allergic asthma was investigated. Methods: (1) purified Derf31 and Der F 1 protein were purified by the purification of prokaryotic expression system through Ni column. (2) cell experiments: Derf31 and DC2.4 incubation flow detection of surface molecular expression; Derf31 and BMDC, splenic cells were incubated to detect IL-4, IFN- gamma secretion, and CFSE method detected cell proliferation. TSLP pathway detection: Derf31 was incubated with normal mice lung tissue cells, A549 were incubated with TLR2 antibody, respectively, and IL-33, TSLP expression was detected by TLR4 inhibitor. (3) animal experiment: using Derf31, Der F 1 to construct allergic asthma model respectively, through serum specific antibody, cytokine, lung pathological section staining. If the model was successful, the ELISA method was used to detect the IL-33 and TSLP content in the lung lavage fluid, the tissue homogenate supernatant, the Siglec-F, CD11c flow antibody labeled eosinophils in the alveolar lavage fluid and the flow cytometry, and the PE-CD90, PerCP-IL-33R, FITC-Lineage and APC-CD45 antibodies were used to mark and detect the ILC2 cells in the lung tissue. At the same time, the total RNA of lung tissue and the content of IL-33, TSLP and IL-13 were detected by RT-PCR. Results: a high concentration of Der F 1 and Derf31 protein.Derf31 could be successfully obtained through the prokaryotic expression system to enhance the CD40 of the DC cell surface molecules, the expression of CD83, and the expression of Derf31, and promote the polarization and proliferation of the cells. After adding TLR2 antibody, the level of TSLP and IL-33 decreased obviously, but the addition of TLR4 inhibitor did not affect the induced allergic asthma in mice successfully, and the level of eosinophil, IL-33 and TSLP increased significantly in group Derf31 and Der F 1, and the expression of IL-33 and TSLP was significantly increased with the blank group. Conclusion: the findings of this study were found by this study. Derf31 can increase the co stimulatory factors on the surface of DC cells and promote the migration of Th0 to Th2; Derf31 can regulate the expression of TSLP and IL-33 in lung epithelial cells by TLR2; in animal experiments, Derf31 can induce obvious eosinophilic allergic asthma and increase the expression of ILC2 cells.
【学位授予单位】：深圳大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25

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