發(fā)布時間：2018-01-24 02:31

  本文關鍵詞： 蛋白質組芯片 血清標志物 結核潛伏感染 活動性結核病 卡介苗接種　出處：《北京市結核病胸部腫瘤研究所》2017年博士論文　論文類型：學位論文

【摘要】：據(jù)統(tǒng)計,全球約三分之一的人感染結核分枝桿菌(Mycobacterium tuberculosis,Mtb),大部分人處于潛伏感染狀態(tài)(Latent tuberculosis infection,LTBI),即宿主和病原體之間保持相對平衡,但當這種平衡一旦被打破,大約5%至10%的人會最終發(fā)展成活動性結核病。因此,及早發(fā)現(xiàn)潛伏感染者,并給予適當?shù)念A防和治療,可以降低結核病的發(fā)病風險,特別是對高危人群的預防和治療更加有意義。目前診斷潛伏感染的方法主要是結核菌素皮膚實驗(Mantoux Tuberculin Skin Test,TST)和γ-干擾素釋放實驗(Interferon-gamma release arrays,IGRAs),而這些方法都存在一定的局限性。在本文第一部分中,我們利用結核分枝桿菌蛋白質組芯片對潛伏感染者和活動性結核病人的血清樣本進行檢測,對芯片結果進行生物信息學分析后,發(fā)現(xiàn)眾多抗原的特異性IgG或IgM抗體的響應程度在兩組人群間具有明顯差異。后續(xù)我們擴大樣本量,選擇差異較大的IgG抗體組的12個抗原,應用酶聯(lián)免疫吸附試驗(Enzyme-linked immunosorbent assay,ELISA)方法進行驗證,受試者操作曲線(Receiver operating characteristic curve,ROC曲線)分析顯示單個抗原Rv2031c和Rv1408在潛伏感染者和活動性結核病患者中具有較好的診斷效果。此外,我們通過二元邏輯回歸分析,建立了一個回歸模型,該模型的診斷效果高于任何一個單一抗原以及Rv2031c、Rv1408和Rv2421c三個抗原的融合效果,其敏感性和特異性均較高,分別為88.04%和96.77%,在利用血清學方法鑒別潛伏感染和活動性結核病方面具有潛在的應用價值�？ń槊�(Bacille Calmette Gurrin,BCG)是世界衛(wèi)生組織(World health organization,WHO)公布的唯一預防結核病的疫苗,據(jù)統(tǒng)計目前累積已有超過30億人口接種過卡介苗。盡管卡介苗的應用如此廣泛,但是其接種后的免疫保護效果的評價一直都沒有一種明確的方法,傳統(tǒng)使用的是觀察BCG接種瘢痕和結核菌素皮膚實驗(TST),這兩種方法都有各自的局限性。同時,對于卡介苗接種后刺激機體產(chǎn)生保護性免疫的具體作用機制仍然有很多未知之處,因此也限制了對疫苗保護效果評價方法的研究。在本研究第二部分中,我們利用結核分枝桿菌蛋白質組芯片,對健康人群和卡介苗成功接種人群的血清樣本進行檢測,結果顯示有142個抗原對IgG抗體有較明顯的差異響應,91個抗原對IgM抗體有較明顯的差異響應,提示這些抗原可引起較強的體液免疫應答,可為深入理解卡介苗的保護機制提供一定的基礎數(shù)據(jù)。后續(xù),我們選擇6個抗原,通過擴大樣本量進行ELISA驗證,結果顯示Rv2728c、Rv2150c和Rv2716抗原在卡介苗接種組中對應的IgG抗體水平明顯高于健康人對照組,ROC分析證實這些抗原均有較好評價效果。二元邏輯回歸分析建立的回歸模型顯示,相比于單個抗原具有更高的敏感性和特異性,分別為95.16%和98.39%,為未來建立一種用來檢測卡介苗接種效果的評價方法奠定基礎。
[Abstract]:According to statistics, about 1/3 people in the world are infected with Mycobacterium tuberculosisus (Mtb). Most people are in latent tuberculosis infection, that is, the relative balance between the host and the pathogen. But when this balance is broken, about 5% to 10% people will eventually develop active TB. Therefore, early detection of latent infections and appropriate prevention and treatment. Can reduce the risk of tuberculosis. Especially for the prevention and treatment of high-risk groups. The current diagnosis of latent infection is mainly tuberculin skin test (. Mantoux Tuberculin Skin Test. TST and Interferon-gamma release arraysme IGR As. In the first part of this paper, we use the proteome chip of Mycobacterium tuberculosis to detect the serum samples of latent and active TB patients. After bioinformatics analysis of the microarray results, it was found that the response of specific IgG or IgM antibodies to many antigens was significantly different between the two groups. Twelve antigens from different IgG antibody groups were selected, and Enzyme-linked immunosorbent assay was used by enzyme-linked immunosorbent assay (Elisa). Erisa method was used to verify the receiver operating characteristic curve. ROC curve) analysis showed that single antigen Rv2031c and Rv1408 had good diagnostic effect in latent infection and active tuberculosis patients. In addition, we used binary logistic regression analysis. A regression model was established and the diagnostic effect of the model was higher than that of any single antigen and the fusion of Rv2031cncnrv1408 and Rv2421c antigens. Its sensitivity and specificity were 88.04% and 96.77%, respectively. It has potential application value in identifying latent infection and active tuberculosis by serological method. Bacille Calmette Gurrin. BCG is the only vaccine against TB published by the World Health Organization (WHO). According to statistics, more than 3 billion people have been vaccinated with BCG vaccine. Although BCG vaccine is widely used, there has been no clear method to evaluate the immune protection effect of BCG vaccine. The traditional method is to observe the scar and tuberculin skin test of BCG, both of which have their own limitations. For the specific mechanism of BCG vaccination to stimulate the body to produce protective immunity, there is still a lot of unknown, so it also limits the study of vaccine protective effect evaluation methods. In the second part of this study. We detected the serum samples of healthy people and successful BCG vaccinated population by using Mycobacterium tuberculosis proteome microarray. The results showed that 142 antigens were significantly different in response to IgG antibodies. The 91 antigens were significantly different in response to IgM antibody, suggesting that these antigens could induce a strong humoral immune response and provide some basic data for further understanding the protective mechanism of BCG vaccine. We selected 6 antigens and verified by ELISA by expanding the sample size. The results showed that Rv2728c. The corresponding IgG antibody levels of Rv2150c and Rv2716 antigens in BCG inoculation group were significantly higher than those in healthy control group. ROC analysis confirmed that these antigens had good evaluation effect. The regression model established by binary logistic regression analysis showed that these antigens were more sensitive and specific than single antigens. It was 95.16% and 98.39, respectively, which laid a foundation for the evaluation of BCG inoculation effect in the future.
【學位授予單位】：北京市結核病胸部腫瘤研究所
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R378.911

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