發(fā)布時間：2018-09-14 07:42

【摘要】：白塞病(Behcet's disease, BD)是一種可能與遺傳因素、環(huán)境微生物感染、以及其他病人生活環(huán)境因素等相關的系統(tǒng)性自身免疫性疾病。目前已有證據(jù)表明該疾病在相對高發(fā)的亞洲人群中具有一些特殊遺傳位點,但與環(huán)境微生物感染方面的相關性仍需要更多的實驗證據(jù)。 本課題采用五種人源細胞系對BD患者的血清自身抗體譜進行測定,通過分析不同細胞免疫熒光強度的差異,選取人臍靜脈內(nèi)皮細胞(HUVEC)為可用于特異性抗原分子篩選的靶細胞。然后分別采用一維分離技術(免疫印跡和免疫沉淀)和二維分離技術(雙向電泳聯(lián)合個體全蛋白質(zhì)組生物芯片)對HUVEC細胞裂解液中的能夠與BD患者血清抗體特異性識別的靶蛋白進行篩選。實驗結果表明,在本課題構建的一維和二維實驗體系中,分別成功篩選出一個新的疾病相關自身抗原。經(jīng)過MALDI-TOF/TOF質(zhì)譜進行序列鑒定,確定其分別為抗增殖蛋白Prohibitin和微管蛋白β-tubulin。然后,通過分子克隆和發(fā)酵表達,分別獲得大量純化的重組蛋白。接著制備對應的酶免檢測試劑盒,進行臨床驗證和疾病癥狀關聯(lián)性分析。結果表明,抗Prohibitin抗體和抗β-tubulin抗體在我國漢族BD患者的血清水平均較健康組有顯著性差異(p0.0001)。 本課題首次在我國漢族BD患者血清中發(fā)現(xiàn)抗Prohibitin抗體和抗β-tubulin抗體的存在,且上述兩種新發(fā)現(xiàn)自身抗體所對應的抗原與微生物熱激蛋白HSP65相關；提示病菌感染、病人的生活環(huán)境等的確都可能參與BD發(fā)病進程,為該疾病的發(fā)病機制和分子診斷研究提供了一定有益信息。
[Abstract]:Behcet's disease (Behcet's disease, BD) is a systemic autoimmune disease that may be associated with genetic factors, environmental microbial infections, and other environmental factors in patients' lives. There is some evidence that the disease has some special genetic loci in the relatively high incidence Asian population, but more experimental evidence is needed for the correlation between the disease and the environmental microorganism infection. In this study, five human cell lines were used to detect the serum autoantibodies of patients with BD. By analyzing the difference of immunofluorescence intensity between different cells, human umbilical vein endothelial cells (HUVEC) were selected as target cells for screening specific antigen molecules. One-dimensional separation (Western blotting and immunoprecipitation) and two-dimensional separation (two-dimensional electrophoresis combined with individual whole proteome biochip) were used to detect specific antibodies in HUVEC cell lysate to patients with BD. Target proteins for sex recognition were screened. The experimental results show that a new disease-related autoantigen was successfully screened out in the one-dimensional and two-dimensional experimental systems constructed in this paper. The antiproliferative protein Prohibitin and tubulin 尾-tubulin were identified by MALDI-TOF/TOF mass spectrometry. Then, a large number of purified recombinant proteins were obtained by molecular cloning and fermentation. Then the corresponding Elisa kit was prepared for clinical verification and disease symptom correlation analysis. The results showed that the serum levels of anti Prohibitin antibody and anti 尾 -tubulin antibody were significantly different from those of healthy controls in Chinese Han nationality patients with BD (p0.0001). In this study, the presence of anti Prohibitin antibody and anti 尾 tubulin antibody was first found in the serum of BD patients of Han nationality in China, and the antigens corresponding to these two new autoantibodies were associated with microbial heat shock protein HSP65. The living environment of the patients may be involved in the pathogenesis of BD, which provides useful information for the pathogenesis and molecular diagnosis of the disease.
【學位授予單位】：北京科技大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R597.9;R446.6

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本文編號：2242027