免疫共沉淀联合质谱分析筛选克罗恩病中与Intelectin-1相互作用蛋白的初步研究

发布时间：2019-05-06 22:44

【摘要】：克罗恩病(Crohn disease,CD)是一种病因复杂、发病机制尚不明确的非特异性炎症性肠道疾病,病变可累及全消化道的任何部位,主要以回盲部病变多见,并伴有肠外表现。克罗恩病的病因复杂,多种致病因素(如感染、环境、自身免疫紊乱及遗传等因素)可能参与其中。近20年来,随着工业化城市进程的发展,克罗恩病在我国发病率逐年升高;因其反复发作,迁延不愈的特点,严重降低了患者的生活质量,引起临床医生的高度重视。肠道粘膜屏障损伤在克罗恩发病机制中,是启动肠道炎症的非常重要的关键环节。凝集素-1(Intelectin-1、ITLN1)是一种主要表达于肺、心脏、小肠及结肠等组织的分泌型糖蛋白,其可参与对抗微生物的肠道免疫防御、胰岛素刺激的葡萄糖摄取、慢性阻塞性肺疾病和哮喘等。我们前期研究发现,相比于克罗恩病病变正常肠粘膜,病变肠粘膜中Intelectin-1蛋白高表达,提示Intelectin-1蛋白可能在克罗恩病发病中起到重要作用。本研究对Intelectin-1相互作用蛋白质进行了分离鉴定,以Intelectin-1特异抗体免疫沉淀分离克罗恩病患者病变肠粘膜组织Intelectin-1蛋白质复合体,SDS-PAGE电泳、染色显影,通过与IgG对照组相比,切取差异性条带行质谱分析,以鉴定Intelectin-1相互作用蛋白,推测在克罗恩病中Intelectin-1及相互作用蛋白作用关系。本研究共鉴定出4个Intelectin-1相互作用蛋白:腺苷酸三磷酸酶(ATPase)、热休克蛋白90(HSP90)、肿瘤坏死因子受体相关因子3(TRAF3)、锌指蛋白(ZNF)。应用免疫共沉淀联合Western blot进一步验证质谱结果。并在克罗恩病病变肠粘膜组织中,鉴定、验证出TRAF3蛋白与Intelectin-1存在相互作用。这些蛋白被认为在克罗恩病的发展中可能发挥重要作用,因此推测Intelectin-1可能通过与它们的相互作用对克罗恩病的发展起到作用。
[Abstract]:Crohn's disease (Crohn disease,CD) is a non-specific inflammatory intestinal disease with complex etiology and unclear pathogenesis. The pathological changes can involve any part of the whole digestive tract, mainly ileocecal lesions, accompanied by extraintestinal manifestations. The etiology of Crohn's disease is complex, and many factors (such as infection, environment, autoimmune disorder and heredity) may be involved in it. In the last 20 years, with the development of industrialized cities, the incidence of Crohn's disease has increased year by year in our country. Because of its repeated attacks and the non-healing characteristics, the quality of life of the patients has been seriously reduced and the clinicians have attached great importance to it. Intestinal mucosal barrier injury plays an important role in the pathogenesis of Crohn's inflammation. Lectin-1 (Intelectin-1,ITLN1) is a secretory glycoprotein mainly expressed in lung, heart, small intestine and colon. It is involved in intestinal immune defense against microorganisms and insulin-stimulated glucose uptake. Chronic obstructive pulmonary disease and asthma, etc. Our previous study found that compared with the normal intestinal mucosa of Crohn's disease, the high expression of Intelectin-1 protein in the pathological intestinal mucosa suggests that Intelectin-1 protein may play an important role in the pathogenesis of Crohn's disease. In this study, Intelectin-1 interacting proteins were isolated and identified. The Intelectin-1 protein complex in intestinal mucosa of patients with Crohn's disease was separated by Intelectin-1 specific antibody immunoprecipitation, and stained by SDS-PAGE electrophoresis. Compared with the IgG control group, the differential bands were cut and analyzed by mass spectrometry to identify the Intelectin-1 interacting proteins. The relationship between the Intelectin-1 and the interacting proteins in Crohn's disease was speculated. Four Intelectin-1 interacting proteins were identified in this study: adenylate triphosphatase (ATPase), heat shock protein 90 (HSP90), tumor necrosis factor receptor related factor 3 (TRAF3) and zinc finger protein (ZNF). The results of mass spectrometry were further verified by immunoprecipitation combined with Western blot. The interaction between TRAF3 protein and Intelectin-1 was identified in intestinal mucosa of Crohn's disease. These proteins are believed to play an important role in the development of Crohn's disease, so we speculate that Intelectin-1 may play an important role in the development of Crohn's disease by interacting with them.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574.62

【参考文献】