本文選題：Derf31 + 過敏性哮喘　； 參考：《深圳大學》2017年碩士論文

【摘要】：背景:近來研究表明,過敏性哮喘的發(fā)生與遺傳、生活方式及所處環(huán)境相關。在眾多因素中,吸入性過敏原是主要的誘因,而且具有區(qū)域差異性。研究發(fā)現(xiàn),在我國塵螨(Dust mite)是誘發(fā)過敏性哮喘的主要因素,如粉塵螨(Dermatophagoides farina)其分泌物、尸體分解物和排泄物等都能誘發(fā)過敏性疾病(蕁麻疹、過敏性鼻炎、哮喘、皮炎等)。有60%成年人哮喘患者對塵螨過敏,在兒童中比例更高,約80%的兒童哮喘患者對塵螨過敏。當過敏原通過呼吸道進入肺部后,肺上皮細胞產(chǎn)生胸腺基質(zhì)淋巴細胞生成素(TSLP)、IL-33,它們可以上調(diào)二型固有淋巴細胞(ILC2s),研究發(fā)現(xiàn)ILC2s與過敏性疾病有著密切的關系。目的:在前期研究中,已成功鑒定出Derf31是粉塵螨新過敏原。本研究通過純化柱富集Derf31蛋白,從細胞實驗和動物實驗兩部分探討Derf31誘發(fā)過敏性哮喘的機制。方法:(1)利用原核表達系統(tǒng)經(jīng)Ni柱純化后獲得較為純凈的Derf31和Der f 1蛋白。(2)細胞實驗:Derf31與DC2.4孵育流式檢測表面分子表達情況;Derf31與BMDC、脾細胞共孵育檢測IL-4、IFN-γ分泌水平,CFSE法檢測細胞增殖情況;TSLP通路檢測:Derf31分別與正常小鼠肺組織細胞、A549共孵育,分別加入TLR2抗體、TLR4抑制劑刺激后檢測IL-33、TSLP表達水平。(3)動物實驗:利用Derf31、Der f 1過敏原分別構(gòu)建過敏性哮喘模型,通過血清特異性抗體、細胞因子、肺部病理切片染色等驗證模型是否成功;通過ELISA法檢測肺部灌洗液、組織勻漿上清中的IL-33和TSLP含量,Siglec-F、CD11c流式抗體標記肺泡灌洗液中嗜酸性粒細胞,流式細胞術檢測其含量;用PE-CD90、PerCP-IL-33R、FITC-Lineage和APC-CD45抗體標記并檢測肺部組織ILC2細胞,同時抽取肺組織總RNA,RT-PCR檢測IL-33、TSLP和IL-13的含量。結(jié)果:通過原核表達系統(tǒng)成功獲得較高濃度的Der f 1和Derf31蛋白。Derf31能夠增強DC細胞表面分子CD40、CD83和CD80的表達,同時促進Th2細胞的極化與增殖。在Derf31與肺上皮細胞共孵育體系中加入TLR2抗體后,TSLP、IL-33水平明顯下降,但加入TLR4抑制劑并沒有影響。Derf31可成功誘發(fā)小鼠過敏性哮喘,并且Derf31組與Der f 1組比較,其嗜酸性粒細胞、IL-33和TSLP水平顯著升高;同時與空白組對比ILC2細胞表達明顯增加。結(jié)論:通過本研究發(fā)現(xiàn),Derf31可上調(diào)DC細胞表面的共刺激因子并促使Th0向Th2偏移;Derf31可通過TLR2調(diào)節(jié)肺部上皮細胞TSLP、IL-33的表達;在動物實驗中,Derf31可誘導明顯的嗜酸性粒細胞型過敏性哮喘,而且使ILC2細胞表達增加。
[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.
【學位授予單位】：深圳大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R562.25

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本文編號：2014614