结核分枝杆菌CE复合物对人单核—巨噬细胞的功能表型的影响及其作用机制

发布时间：2018-05-29 07:44

本文选题：结核分枝杆菌 + CE复合物　；参考：《安徽医科大学》2008年硕士论文

【摘要】： 结核分枝杆菌感染所致的结核病仍然是传染病中引起死亡的首位疾病,全球近三分之一的人口感染结核分枝杆菌,每年有超过800万新增结核感染者,至少有200万人死于结核病。推测发展成活动性TB的个体既可能是结核杆菌短期新发感染也可能是长期潜伏后活动所致,这种宿主与病原体之间的相互作用可能决定了不同的感染结果。结核杆菌是典型的胞内致病菌,它在机体内的主要宿主细胞是巨噬细胞。结核杆菌与巨噬细胞膜表面受体结合后,经膜内陷形成吞噬小体进入细胞。巨噬细胞会通过吞噬体溶酶体融合,产生大量代谢性活性由基如反应性氮中间产物和反应性氧中间产物,自身凋亡,分泌大量细胞因子发挥免疫调节作用以及加工提呈结核杆菌抗原给CD4+T和CD8+T细胞,产生结核杆菌抗原特异性的T细胞等途径对结核杆菌进行杀伤和清除。结核杆菌也会通过干扰上述途径来逃避杀伤和清除。结核杆菌在感染或者生长过程中,不同阶段会产生出大量蛋白,如在早期感染或者早期培养阶段产生一些低分子量抗原如ESAT-6,CFP-10等,生长阶段会分泌Ag85复合物,MPT53,MPT63,MPT64,MPT70,MPT32,38kd,19kd及LAM,PGL等细胞壁和胞质抗原。这些抗原多能诱导强烈的细胞免疫反应,而一些则与结核杆菌免疫调节以及免疫逃避有关。其中CFP-10和ESAT-6由只出现在致病结核分枝杆菌中的RD1区基因编码产生,具有很强的特异性,BCG以及非致病性分枝杆菌缺乏,二者在体内按1:1紧密连接形成一个杂二聚体的复合物发挥作用。ESAT-6和CFP-10分别都能激活巨噬细胞产生TNF-α和NO以及表达抗原提呈相关功能分子如B7.1(CD80), MHC-II,ICAM-I等;它们都能强烈诱导小鼠骨髓细胞分化为树突状细胞样的抗原提呈细胞。做为一个紧密连接的整体,CE复合物可能以CFP-10蛋白C端与人单核细胞表面结合,诱导相应的细胞信号通路变化,从而调变宿主单核-巨噬细胞的抗结核免疫反应。【目的】研究结核分枝杆菌CE复合物对人单核-巨噬细胞功能表型的影响及其作用的细胞信号传导通路,从而发现结核分枝杆菌逃避巨噬细胞杀伤,造成潜伏以及慢性感染的可能机制,为结核病的防治提供新的思路。【方法】用大肠杆菌克隆表达纯化的CE复合物与单核细胞系THP-1细胞或者人血来源的单核细胞共同培养,分别设定不同浓度的CE复合物以及不同培养时间,收集细胞培养上清以及细胞。采用ELISA法检测CE复合物刺激人单核-巨噬细胞TNF-α、IL-10、IL-12的产生的水平,采用流式细胞仪检测CE复合物刺激细胞表型的变化;并且应用各种细胞信号传导通路抑制剂来发现CE复合物作用的细胞信号传导通路。【结果】结核分枝杆菌CE复合物能够在共同培养早期(4～8h)诱导人单核-巨噬细胞活化,大量产生TNF-α;细胞信号传导通路MEK1/2和P38 MAPK的选择性抑制剂SB203580,PD98059,U0126能抑制CE的这种作用;CE复合物在早期(18h)促进单核-巨噬细胞表面抗原提呈功能分子HLA-DR,CD80,CD40的表达。但在同培养晚期(48h),CE复合物则抑制单核巨噬细胞TNF-α的产生,抑制抗原提呈功能分子HLA-DR,CD80,CD40的表达,促进IL-10的产生,以及在IL-4的参与下刺激单核细胞表面大量表达树突状细胞特异性ICAM-3捕获非整合素(DC-SIGN)。【结论】CE复合物在感染早期可能通过激活细胞信号传导通路MEK1/2和P38 MAPK刺激单核巨噬细胞活化,形成强烈的抗结核免疫反应,限制结核杆菌在体内的生存,可能具有重要的保护意义;但在感染晚期,CE复合物则可能抑制单核巨噬细胞对结核分枝杆菌感染的保护性细胞免疫反应,抑制单核巨噬细胞对结核分枝杆菌的杀伤作用,帮助结核分枝杆菌在单核巨噬细胞内生存,造成潜伏以及慢性感染。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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