免疫共沉淀聯(lián)合質(zhì)譜分析篩選克羅恩病中與Intelectin-1相互作用蛋白的初步研究
發(fā)布時(shí)間：2019-05-06 22:44
【摘要】：克羅恩病(Crohn disease,CD)是一種病因復(fù)雜、發(fā)病機(jī)制尚不明確的非特異性炎癥性腸道疾病,病變可累及全消化道的任何部位,主要以回盲部病變多見,并伴有腸外表現(xiàn)。克羅恩病的病因復(fù)雜,多種致病因素(如感染、環(huán)境、自身免疫紊亂及遺傳等因素)可能參與其中。近20年來,隨著工業(yè)化城市進(jìn)程的發(fā)展,克羅恩病在我國(guó)發(fā)病率逐年升高;因其反復(fù)發(fā)作,遷延不愈的特點(diǎn),嚴(yán)重降低了患者的生活質(zhì)量,引起臨床醫(yī)生的高度重視。腸道粘膜屏障損傷在克羅恩發(fā)病機(jī)制中,是啟動(dòng)腸道炎癥的非常重要的關(guān)鍵環(huán)節(jié)。凝集素-1(Intelectin-1、ITLN1)是一種主要表達(dá)于肺、心臟、小腸及結(jié)腸等組織的分泌型糖蛋白,其可參與對(duì)抗微生物的腸道免疫防御、胰島素刺激的葡萄糖攝取、慢性阻塞性肺疾病和哮喘等。我們前期研究發(fā)現(xiàn),相比于克羅恩病病變正常腸粘膜,病變腸粘膜中Intelectin-1蛋白高表達(dá),提示Intelectin-1蛋白可能在克羅恩病發(fā)病中起到重要作用。本研究對(duì)Intelectin-1相互作用蛋白質(zhì)進(jìn)行了分離鑒定,以Intelectin-1特異抗體免疫沉淀分離克羅恩病患者病變腸粘膜組織Intelectin-1蛋白質(zhì)復(fù)合體,SDS-PAGE電泳、染色顯影,通過與IgG對(duì)照組相比,切取差異性條帶行質(zhì)譜分析,以鑒定Intelectin-1相互作用蛋白,推測(cè)在克羅恩病中Intelectin-1及相互作用蛋白作用關(guān)系。本研究共鑒定出4個(gè)Intelectin-1相互作用蛋白:腺苷酸三磷酸酶(ATPase)、熱休克蛋白90(HSP90)、腫瘤壞死因子受體相關(guān)因子3(TRAF3)、鋅指蛋白(ZNF)。應(yīng)用免疫共沉淀聯(lián)合Western blot進(jìn)一步驗(yàn)證質(zhì)譜結(jié)果。并在克羅恩病病變腸粘膜組織中,鑒定、驗(yàn)證出TRAF3蛋白與Intelectin-1存在相互作用。這些蛋白被認(rèn)為在克羅恩病的發(fā)展中可能發(fā)揮重要作用,因此推測(cè)Intelectin-1可能通過與它們的相互作用對(duì)克羅恩病的發(fā)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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574.62
本文編號(hào)：2470552
[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.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R574.62
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相關(guān)期刊論文 前3條
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