结核分枝杆菌—巨噬细胞相互作用的研究

发布时间：2018-04-25 18:04

本文选题：结核分枝杆菌 + 趋化因子和趋化因子受体　；参考：《复旦大学》2005年博士论文

【摘要】：结核分枝杆菌(Mycobacterium tuberculosis)是已经发现的最为重要的传染性病原体之一，每年可以引起2—3百万人感染结核病。结核分枝杆菌是典型的胞内致病菌，它可以在宿主巨噬细胞内存活。巨噬细胞和结核分枝杆菌的初始相互作用中免疫调节因子(包括细胞因子、趋化因子及其它们的受体)，，在细菌能否引起机体发病中可能起着关键作用。目前，对于结核杆菌感染中趋化因子和趋化因子受体的了解十分有限。为了弄清楚趋化因子和趋化因子受体在结核杆菌感染的巨噬细胞中的变化，我们利用活化的人巨噬细胞系U937作为结核杆菌感染的体外模型，应用基因芯片检测技术、半定量PCR、流式细胞术等手段来研究巨噬细胞在不同感染阶段(0小时，6小时，12小时和24小时)的趋化因子和趋化因子受体的表达谱。基因芯片的结果显示，结核分枝杆菌H37Rv感染巨噬细胞6小时和12小时都可以检测到有变化的基因，感染24小时，明显上调的基因有25条，其中显著上调的基因有22条。半定量RT—PCR证实了部分基因(IL-8，MIP1-α，RANTES，CXCR4和CCR5)的芯片检测结果。我们在用基因芯片法检测了结核杆菌感染巨噬细胞后的全部趋化因子和趋化因子受体的表达变化中，发现两个重要的趋化因子受体CCR5和CXCR4是表达上调的，CCR5和CXCR4是人类艾滋病病毒HIV入侵人体细胞的两个重要辅助受体，结合我们在临床上观察到的有一部分的艾滋病患者同时患有结核病，我们推测趋化因子受体CCR5和CXCR4在结核分枝杆菌感染后的表达上调可能是结核病／艾滋病共患的另一个原因。为了了解CCR5和CXCR4的上调表达是结核杆菌感染特异产生的还是炎症类致病菌都能产生的普遍现象，我们又用另一种致病菌伴放线放线杆菌(Actinobacillus actinomycetemcomitans)去攻击巨噬细胞，然后从mRNA和蛋白水平观测CCR5和CXCR4的表达变化，结果表明伴放线放线杆菌也能刺激这两个分子的上调表达。为了进一步揭示CCR5和CXCR4表达变化的分子机理，研究了结核分枝杆菌感染后巨噬细胞信号转导通路的变化，详细考察了CCR5和CXCR4的启动子结构，然后我们定位在MAPK通路和共同的调节因子YY1上。运用p38 MAPK的特异性抑制剂SB203580，我们研究其对CCR5，CXCR4和CCR5、CXCR4的共同的负调控转录因子YY1的表达影响。最终的实验结果表明在我们的模型中 CCR5和CXCR4的上调主要是p38 MAPK介导的，YY1依赖，这个结果表明CCR5和CXCR4的上调和MTB／HIV共感染之间的几乎没有关联。
[Abstract]:Mycobacterium tuberculosisis is one of the most important infectious pathogens which can cause 2 to 3 million people to become infected with tuberculosis every year. Mycobacterium tuberculosis is a typical intracellular pathogen that can survive in host macrophages. Immunomodulatory factors (including cytokines, chemokines and their receptors) play a key role in the initial interaction between macrophages and Mycobacterium tuberculosis. At present, the understanding of chemokines and chemokine receptors in Mycobacterium tuberculosis infection is very limited. In order to find out the changes of chemokines and chemokine receptors in macrophages infected by Mycobacterium tuberculosis, we used activated human macrophage cell line U937 as an in vitro model of Mycobacterium tuberculosis infection. Semi-quantitative PCR and flow cytometry were used to study the expression profiles of chemokines and chemokine receptors in macrophages at different stages of infection. The results of microarray showed that the mutated genes could be detected in macrophages infected with Mycobacterium tuberculosis H37Rv for 6 hours and 12 hours. After 24 hours of infection, 25 genes were obviously up-regulated, among which 22 genes were significantly up-regulated. Semi-quantitative RT-PCR confirmed the microarray detection results of IL-8 mil MIP1- 伪 RANTESX CR4 and CCR5. We detected the expression of all chemokines and chemokine receptors in macrophages infected with Mycobacterium tuberculosis by gene chip method. Two important chemokine receptors, CCR5 and CXCR4, were found to be two important coreceptors for the invasion of human cells by HIV / HIV. We speculate that the up-regulated expression of chemokine receptors CCR5 and CXCR4 after Mycobacterium tuberculosis infection may be another cause of TB / AIDS co-infection. To understand whether the upregulation of CCR5 and CXCR4 is specific to Mycobacterium tuberculosis infection or a common form of inflammatory pathogens, we used another pathogen, Actinobacillus actinomycetemcomitans., to attack macrophages. The expression of CCR5 and CXCR4 was observed at the level of mRNA and protein. The results showed that Actinobacillus actinomycetes could also stimulate the up-regulation of the expression of these two molecules. In order to further reveal the molecular mechanism of the expression of CCR5 and CXCR4, the changes of macrophage signal transduction pathway after Mycobacterium tuberculosis infection were studied, and the promoter structures of CCR5 and CXCR4 were investigated in detail. We then localize the MAPK pathway and the common regulatory factor YY1. Using SB203580, a specific inhibitor of p38 MAPK, we studied the effects of SB203580 on the expression of YY1, a common negative regulatory transcription factor, of CCR5CXCR4 and CCR5CXCR4. The final experimental results show that in our model, The up-regulation of CCR5 and CXCR4 was mainly mediated by p38 MAPK, which indicated that there was almost no association between the up-regulation of CCR5 and CXCR4 and MTB/HIV co-infection.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R378

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