本文選題：結(jié)核分枝桿菌 + CE復(fù)合物��； 參考：《安徽醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 結(jié)核分枝桿菌感染所致的結(jié)核病仍然是傳染病中引起死亡的首位疾病,全球近三分之一的人口感染結(jié)核分枝桿菌,每年有超過(guò)800萬(wàn)新增結(jié)核感染者,至少有200萬(wàn)人死于結(jié)核病。推測(cè)發(fā)展成活動(dòng)性TB的個(gè)體既可能是結(jié)核桿菌短期新發(fā)感染也可能是長(zhǎng)期潛伏后活動(dòng)所致,這種宿主與病原體之間的相互作用可能決定了不同的感染結(jié)果。結(jié)核桿菌是典型的胞內(nèi)致病菌,它在機(jī)體內(nèi)的主要宿主細(xì)胞是巨噬細(xì)胞。結(jié)核桿菌與巨噬細(xì)胞膜表面受體結(jié)合后,經(jīng)膜內(nèi)陷形成吞噬小體進(jìn)入細(xì)胞。巨噬細(xì)胞會(huì)通過(guò)吞噬體溶酶體融合,產(chǎn)生大量代謝性活性由基如反應(yīng)性氮中間產(chǎn)物和反應(yīng)性氧中間產(chǎn)物,自身凋亡,分泌大量細(xì)胞因子發(fā)揮免疫調(diào)節(jié)作用以及加工提呈結(jié)核桿菌抗原給CD4+T和CD8+T細(xì)胞,產(chǎn)生結(jié)核桿菌抗原特異性的T細(xì)胞等途徑對(duì)結(jié)核桿菌進(jìn)行殺傷和清除。結(jié)核桿菌也會(huì)通過(guò)干擾上述途徑來(lái)逃避殺傷和清除。結(jié)核桿菌在感染或者生長(zhǎng)過(guò)程中,不同階段會(huì)產(chǎn)生出大量蛋白,如在早期感染或者早期培養(yǎng)階段產(chǎn)生一些低分子量抗原如ESAT-6,CFP-10等,生長(zhǎng)階段會(huì)分泌Ag85復(fù)合物,MPT53,MPT63,MPT64,MPT70,MPT32,38kd,19kd及LAM,PGL等細(xì)胞壁和胞質(zhì)抗原。這些抗原多能誘導(dǎo)強(qiáng)烈的細(xì)胞免疫反應(yīng),而一些則與結(jié)核桿菌免疫調(diào)節(jié)以及免疫逃避有關(guān)。其中CFP-10和ESAT-6由只出現(xiàn)在致病結(jié)核分枝桿菌中的RD1區(qū)基因編碼產(chǎn)生,具有很強(qiáng)的特異性,BCG以及非致病性分枝桿菌缺乏,二者在體內(nèi)按1:1緊密連接形成一個(gè)雜二聚體的復(fù)合物發(fā)揮作用。ESAT-6和CFP-10分別都能激活巨噬細(xì)胞產(chǎn)生TNF-α和NO以及表達(dá)抗原提呈相關(guān)功能分子如B7.1(CD80), MHC-II,ICAM-I等;它們都能強(qiáng)烈誘導(dǎo)小鼠骨髓細(xì)胞分化為樹突狀細(xì)胞樣的抗原提呈細(xì)胞。做為一個(gè)緊密連接的整體,CE復(fù)合物可能以CFP-10蛋白C端與人單核細(xì)胞表面結(jié)合,誘導(dǎo)相應(yīng)的細(xì)胞信號(hào)通路變化,從而調(diào)變宿主單核-巨噬細(xì)胞的抗結(jié)核免疫反應(yīng)。 【目的】研究結(jié)核分枝桿菌CE復(fù)合物對(duì)人單核-巨噬細(xì)胞功能表型的影響及其作用的細(xì)胞信號(hào)傳導(dǎo)通路,從而發(fā)現(xiàn)結(jié)核分枝桿菌逃避巨噬細(xì)胞殺傷,造成潛伏以及慢性感染的可能機(jī)制,為結(jié)核病的防治提供新的思路。 【方法】用大腸桿菌克隆表達(dá)純化的CE復(fù)合物與單核細(xì)胞系THP-1細(xì)胞或者人血來(lái)源的單核細(xì)胞共同培養(yǎng),分別設(shè)定不同濃度的CE復(fù)合物以及不同培養(yǎng)時(shí)間,收集細(xì)胞培養(yǎng)上清以及細(xì)胞。采用ELISA法檢測(cè)CE復(fù)合物刺激人單核-巨噬細(xì)胞TNF-α、IL-10、IL-12的產(chǎn)生的水平,采用流式細(xì)胞儀檢測(cè)CE復(fù)合物刺激細(xì)胞表型的變化;并且應(yīng)用各種細(xì)胞信號(hào)傳導(dǎo)通路抑制劑來(lái)發(fā)現(xiàn)CE復(fù)合物作用的細(xì)胞信號(hào)傳導(dǎo)通路。 【結(jié)果】結(jié)核分枝桿菌CE復(fù)合物能夠在共同培養(yǎng)早期(4～8h)誘導(dǎo)人單核-巨噬細(xì)胞活化,大量產(chǎn)生TNF-α;細(xì)胞信號(hào)傳導(dǎo)通路MEK1/2和P38 MAPK的選擇性抑制劑SB203580,PD98059,U0126能抑制CE的這種作用;CE復(fù)合物在早期(18h)促進(jìn)單核-巨噬細(xì)胞表面抗原提呈功能分子HLA-DR,CD80,CD40的表達(dá)。但在同培養(yǎng)晚期(48h),CE復(fù)合物則抑制單核巨噬細(xì)胞TNF-α的產(chǎn)生,抑制抗原提呈功能分子HLA-DR,CD80,CD40的表達(dá),促進(jìn)IL-10的產(chǎn)生,以及在IL-4的參與下刺激單核細(xì)胞表面大量表達(dá)樹突狀細(xì)胞特異性ICAM-3捕獲非整合素(DC-SIGN)。 【結(jié)論】CE復(fù)合物在感染早期可能通過(guò)激活細(xì)胞信號(hào)傳導(dǎo)通路MEK1/2和P38 MAPK刺激單核巨噬細(xì)胞活化,形成強(qiáng)烈的抗結(jié)核免疫反應(yīng),限制結(jié)核桿菌在體內(nèi)的生存,可能具有重要的保護(hù)意義;但在感染晚期,CE復(fù)合物則可能抑制單核巨噬細(xì)胞對(duì)結(jié)核分枝桿菌感染的保護(hù)性細(xì)胞免疫反應(yīng),抑制單核巨噬細(xì)胞對(duì)結(jié)核分枝桿菌的殺傷作用,幫助結(jié)核分枝桿菌在單核巨噬細(xì)胞內(nèi)生存,造成潛伏以及慢性感染。
[Abstract]:Tuberculosis caused by Mycobacterium tuberculosis is still the first disease that causes death in infectious diseases. Nearly 1/3 of the world's population is infected with Mycobacterium tuberculosis, with more than 8 million new TB infections each year, and at least 2 million people die from tuberculosis. It is presumed that the individual developing active TB may be a short-term new sense of tuberculosis. The infection may also be caused by long-term latent activity. The interaction between the host and the pathogen may determine the result of different infection. The Mycobacterium tuberculosis is a typical intracellular pathogen, and the main host cell in the body is macrophage. After combining with the membrane surface receptor of the macrophage, the Mycobacterium tuberculosis is formed to form phagocytic bodies. The macrophages can be fused by the phagocytic lysosome to produce a large number of metabolic activities, such as reactive nitrogen intermediate products and reactive oxygen intermediate products, and their own apoptosis, secreting a large number of cytokines to play the role of immunomodulatory function and the processing of Mycobacterium tuberculosis antigen to CD4+T and CD8+T cells to produce the specific antigen specific antigen of Mycobacterium tuberculosis The Mycobacterium tuberculosis can also be killed and removed by means of sex T cells. The Mycobacterium tuberculosis also avoids killing and removing by interfering with the above pathway. In the process of infection or growth, the Mycobacterium tuberculosis produces a large number of proteins in different stages, such as producing some low molecular weight antigens such as ESAT-6, CFP-10, etc. in early infection or early culture stage. The growth phase secretes Ag85 complexes, MPT53, MPT63, MPT64, MPT70, MPT32,38kd, 19kd and LAM, PGL and other cell walls and cytoplasmic antigens. These antigens can induce a strong cellular immune response, and some are related to immunoregulation and immune escape from Mycobacterium tuberculosis. CFP-10 and ESAT-6 are RD1 only in the pathogenesis of Mycobacterium tuberculosis. The gene encoding of the region is highly specific, BCG and non pathogenic mycobacteria lack. The two compounds are closely connected by 1:1 to form a hybrid two polymer in the body to play the role of.ESAT-6 and CFP-10 to activate macrophages to produce TNF- alpha and NO, as well as the expression of antigen related functional molecules such as B7.1 (CD80), MHC-II, ICAM. -I and so on; they all strongly induce mouse bone marrow cells to differentiate into dendritic cells like antigen presenting cells. As a tightly connected whole, the CE complex may be combined with the surface of human mononuclear cells on the CFP-10 protein C end, inducing the corresponding cell signaling pathway changes, thus regulating the anti tuberculosis immune response of the host monocyte macrophage.
[Objective] to study the effect of Mycobacterium tuberculosis CE complex on the functional phenotype of human mononuclear macrophage and its cellular signal transduction pathway, and to find out the possible mechanism of Mycobacterium tuberculosis to escape the killing of macrophages, cause latent and chronic infection, and provide new ideas for the prevention and control of tuberculosis.
[Methods] the purified CE complex was co cultured with mononuclear cell line THP-1 cells or human mononuclear cells from human blood. The CE complexes of different concentrations and different culture time were set respectively, and cell culture supernatant and cells were collected. ELISA method was used to detect CE complex to stimulate human mononuclear macrophage TNF- alpha. At the level of IL-10, IL-12, a flow cytometer was used to detect the changes in the phenotype of the CE complex, and a variety of cell signaling pathway inhibitors were used to detect the cell signaling pathway of the CE complex.
[results] Mycobacterium tuberculosis CE complex can induce human mononuclear macrophage activation in the early stage of co culture (4 ~ 8h) and produce a large number of TNF- alpha; the selective inhibitors of MEK1/2 and P38 MAPK, SB203580, PD98059, and U0126, can inhibit the effect of CE, and CE complex in the early stage (18h) promotes the surface of mononuclear macrophage. Antigen presentation of functional molecules HLA-DR, CD80, CD40 expression. But in the same period of culture (48h), CE complex inhibits the production of mononuclear macrophage TNF- alpha, inhibits the expression of functional molecules HLA-DR, CD80, CD40, promotes the production of IL-10, and stimulates a large number of dendritic cell specific ICAM-3 to stimulate the surface of mononuclear cells under the participation of IL-4. Non integrin (DC-SIGN) was captured.
[Conclusion] CE complex may stimulate mononuclear macrophage activation by activating cell signal transduction pathway MEK1/2 and P38 MAPK in early infection, forming a strong anti tuberculosis immune response and restricting the survival of Mycobacterium tuberculosis in the body, which may have important protective significance. But in the late infection, CE complex may inhibit mononuclear macrophage. The protective cell immune response to Mycobacterium tuberculosis infection inhibits the killing effect of mononuclear macrophages on Mycobacterium tuberculosis, helps Mycobacterium tuberculosis survive in mononuclear macrophages, and causes latent and chronic infection.
【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】：2008
【分類號(hào)】：R392

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