白三烯B4在巨噬细胞抗结核免疫中的作用及机制研究

发布时间：2018-01-08 21:28

本文关键词：白三烯B4在巨噬细胞抗结核免疫中的作用及机制研究　出处：《第四军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：结核病(TB)仍然是全球范围公共健康问题,主要由结核分枝杆菌(Mtb)感染引起,Mtb是一种胞内寄生菌,感染人体后主要居住在巨噬细胞内,巨噬细胞作为吞噬Mtb的主要细胞在感染Mtb的机体固有免疫应答中起主要作用,构成机体防御的第一道防线,该细胞主要通过吞噬、生成自由基及多种细胞因子、细胞凋亡、抗原提呈等多种途径杀灭Mtb。宿主抗Mtb感染的免疫机制复杂,众多免疫相关的介质参与其中,巨噬细胞是白三烯B4(LTB4)的主要来源之一,白三烯是花生四烯酸通过5-脂氧合酶途径代谢的产物,是有效的促炎脂质介质,研究表明LTB4具有多重功能,可导致白细胞趋化、粘附、脱颗粒、细胞因子分泌,可以影响多种生物效应,参与许多免疫性疾病的病理过程,如哮喘、类风湿性关节炎等,在疾病的病理过程中发挥重要作用。近年来LTB4在感染性疾病发挥的作用日益受到重视,包括寄生虫、细菌感染,但目前有关LTB4在结核感染中发挥的作用少有报道。本研究提出旨在探索如下问题:结核感染是否促进巨噬细胞表达LTB4,白三烯B4对巨噬细胞抗结核免疫中发挥的作用以及可能的机制,期望为临床抗结核病的辅助治疗提供实验依据。实验一:结核分枝杆菌及其抗原成份对巨噬细胞LTB4表达的影响目的:了解结核感染中引起巨噬细胞表达LTB4的成分。方法:采用不同剂量结核重组抗原Ag85B、ESAT-6和菌株H37Ra及BCG与巨噬细胞共培养4、24h,留取培养上清,ELISA检测LTB4表达量。结果:在共培养4h、24h时间段,巨噬细胞在结核及其抗原的刺激下均能生成LTB4,除H37Ra组外实验组与对照组比较均有统计学差异,且LTB4的量分别随Ag85B,ESAT-6量增多而增多,培养24h各组生成的白三烯B4量少于4h。结论:BCG和结核分支杆菌分泌蛋白ESAT-6、Ag85B促进巨噬细胞早期表达LTB4,而热灭活H37Ra不能促进巨噬细胞表达LTB4。实验二:LTB4对巨噬细胞抗结核免疫的作用目的:探索LTB4在巨噬细胞感染Mtb过程中发挥的作用。方法:采用改良抗酸染色方法确定THP-1吞噬现象,通过流式细胞术定量检测吞噬率;流式检测LTB4对感染细胞活性氧的影响;酸性磷酸酶染色确定LTB4对溶酶体影响;流式检测感染细胞凋亡率。结果:(1)比较加入LTB4后THP-1细胞对BCG的吞噬变化,在培养4h不同浓度外源性LTB4(1-100nM)都可以促进吞噬,吞噬率随剂量升高而升高,且这种作用可以被LTB4的BLT1受体阻滞剂U75302抑制,培养24h无促吞噬作用。(3)LTB4可以促进细胞活性氧的产生,并且可以提高感染细胞的活性氧。(4)LTB4促进感染细胞凋亡。结论:白三烯B4可以促进THP-1细胞吞噬Mtb,并通过产生活性氧和促进细胞凋亡来影响巨噬细胞抗结核免疫作用。实验三:探索LTB4对巨噬细胞抗结核免疫作用的机制目的:探索LTB4影响巨噬细胞抗结核免疫可能的机制。方法:巨噬细胞和结核共培养4、24h,施加不同的干预措施,采用qPCR和酶联免疫吸附试验测定感染结核的巨噬细胞TNF-α的mRNA及培养上清蛋白水平的表达,以及LTB4生成关键酶LTA4H的mRNA及LTB4表达水平。结果:感染4h、24h,外源性LTB4对感染结核巨噬细胞TNF-α表达水平均无影响,但促进其mRNA表达,4h后LTA4H生成抑制剂Bestatin下调TNF-α的mRNA水平,24h后LTB4受体抑制剂U75302下调TNF-α的mRNA水平。结论:外源性LTB4不影响TNF-α表达,但增高其mRNA水平,应用LTB4抑制剂可部分下调TNF-α的mRNA表达水平。
[Abstract]:Tuberculosis (TB) is still a global public health problem, mainly by Mycobacterium tuberculosis (Mtb) infection, Mtb is an intracellular pathogen infection in the human body, mainly living in macrophages and macrophage phagocytosis of Mtb cells as the main body of the Mtb infection in the solid have played a major role in immune response, constitute the first line of defense for the body of the cell, mainly through phagocytosis, generation of free radicals and cytokines, apoptosis, immune mechanism of antigen presenting a variety of ways such as killing Mtb. host anti Mtb infection complicated, many immune related mediators involved, macrophage leukotriene B4 (LTB4) is one of the main sources, is a product of leukotrienes four arachidonic acid by 5- lipoxygenase pathway, proinflammatory lipid mediators is effective, the results show that LTB4 has multiple functions, can lead to leukocyte chemotaxis, adhesion, degranulation, cytokine Secretion, can affect a variety of biological effects, pathological processes involved in many autoimmune diseases such as asthma, rheumatoid arthritis, play an important role in the pathological process of the disease. In recent years LTB4 in infectious diseases play increasingly important role, including parasites, bacterial infection, but the LTB4 in tuberculosis the role is rarely reported. This study aims to explore the following questions: whether tuberculosis infection promotes macrophage LTB4 expression play leukotriene B4 on macrophages anti TB immune function and the possible mechanism, to provide experimental basis for clinical adjuvant therapy of anti tuberculosis expectations. Experiment one: effects of Mycobacterium tuberculosis and its antigens expressed on macrophages Objective: To investigate the LTB4 tuberculosis infection caused by macrophages expressing LTB4 components. Methods: using different doses of tuberculosis recombinant antigen Ag85B, ESAT 4,24h and -6 co cultured strains H37Ra and BCG with macrophages, collected supernatant, detect the expression of ELISA LTB4. Results: in co culture 4h, 24h period, LTB4 in macrophages can produce tuberculosis and antigen stimulation, except group H37Ra experimental group compared with the control group had statistically significant difference, and the amount of LTB4 respectively with Ag85B, ESAT-6 increased and increased the amount of 24h training of leukotriene B4 generation were less than 4h. conclusion: the secreted protein ESAT-6 of Mycobacterium tuberculosis BCG and Ag85B, promote the expression of LTB4 of macrophages, and heat inactivated H37Ra can promote the expression of LTB4. in experiment two macrophages: effect of LTB4 on macrophages anti tuberculosis immunity objective: To explore the role LTB4 play in the infection of macrophages in the process of Mtb. Methods: using the modified acid fast staining method to determine THP-1 phagocytosis, by flow cytometry quantitative detection of phagocytic rate; flow cytometry detection of LTB4 infection on fine Influence of intracellular reactive oxygen species; acid phosphatase staining to determine the effect of LTB4 on lysosomes; flow cytometry infected cells apoptosis rate. Results: (1) comparison with changes in phagocytosis of THP-1 cells to BCG LTB4 after 4H in culture, different concentrations of exogenous LTB4 (1-100nM) can promote the phagocytosis, phagocytosis rate with the dose increased. And this effect can be BLT1 receptor antagonist U75302 LTB4 inhibition, 24h culture without promoting phagocytosis. (3) LTB4 cells can promote the generation of reactive oxygen species and oxygen can increase the activity of infected cells. (4) LTB4 infection promote cell apoptosis. Conclusion: leukotriene B4 can promote THP-1 cell phagocytosis of Mtb, and the the production of reactive oxygen species and promoting apoptosis effect of macrophages anti tuberculosis immunity. Experiment three: Objective To explore the mechanism of LTB4 on macrophage anti tuberculosis immune function: To explore the mechanism of LTB4 on macrophages anti tuberculosis immunity may be. Methods: 4,24h were co cultured with macrophages and tuberculosis, applying different interventions, the supernatant protein level determination of tuberculosis infection of macrophages TNF- alpha by qPCR and enzyme-linked immunosorbent assay mRNA and culture, and the expression of mRNA and LTB4 LTA4H key enzyme generated LTB4 level. Results: 4H infection, 24h, exogenous LTB4 influence on the level of tuberculosis infection of macrophage TNF- expression, but the level of mRNA expression, 4h LTA4H inhibitor Bestatin reduced TNF- alpha mRNA levels, 24h LTB4 receptor inhibitor U75302 down-regulation of TNF- alpha mRNA levels. Conclusion: the exogenous LTB4 does not affect TNF- expression, but increased the level of mRNA, application of LTB4 inhibitor can be part of the down-regulation of TNF- expression level of mRNA alpha.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R52

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