不可分型流感嗜血杆菌在呼吸道感染中的免疫应答对细胞内受体TLR9在感染中的表达研究

发布时间：2018-01-06 07:13

  本文关键词：不可分型流感嗜血杆菌在呼吸道感染中的免疫应答对细胞内受体TLR9在感染中的表达研究　出处：《浙江大学》2012年博士论文　论文类型：学位论文

  更多相关文章： 流感嗜血杆菌 呼吸道感染 细胞内受体 TLR9

【摘要】：高级脊椎动物发展了两套保护性免疫系统,天然免疫和获得性免疫。天然免疫系统主要依赖于通过感知病原体相关分子模式(pathogen-associated molecular patterns, PAMPs)的蛋白质识别病原体。TLRs (toll-like receptors)是研究较多的模式识别受体,TLR识别一系列的细菌、真菌及病毒产物,TLR9与其他LTRs不同,存在于细胞内,识别细胞内非甲基化细菌DNA。 呼吸道感染与不可分型流感嗜血杆菌(non-typeable Haemophilus Influenza, NHTi)具有相关性,特别是肺部及中耳细菌感染,具有一定的发病率及死亡率。NTHi是一种无荚膜的革兰氏阴性多形杆菌,常定植于大多数人的上气道,在机体气道表面受损或在粘液纤毛清除功能散失时常常引起机会性感染的病原体。NTHi传统意义上被认为是一种非侵入性的细胞外细菌,但近期研究表明,NTHi可内化至真核细胞内,侵入上皮细胞及巨噬细胞并在细胞内生存,从而躲避抗菌素的杀菌活性。 我们应用抗菌素保护策略,将细胞同NTHi共孵育2h后；通过高清显微镜直观的观察NTHi进入到细胞内,NTHi可在胞外产生炎症反应的同时,内化入细胞内,故NTHi同时作为兼性胞内菌存在；同时,我们构建了急性肺部NTHi感染模型,应用原位杂交技术,在NTHi刺激ALIM4h后,在肺泡腔内可检测到NTHi的存在。 应用APA策略,定量检测到NTHi刺激后人上皮细胞A549及外周血单核细胞及肺组织后产生的炎症因子IL-8和／或TNF-a水平,总炎症因子与胞内NTHi产生的炎症因子比较,胞内菌炎症因子处于非优势地位。ALIM经NTHi刺激后有炎症因子的释放。活性NTHi所产生炎症效应同灭活菌比较无显著差异；细菌成分如细菌DNA,可诱导细胞产生炎症介质,此作用可被核内体成熟抑制剂氯喹阻断。 将活性NTHi、灭活的NTHi及细菌DNA刺激上皮细胞A549及外周血单核细胞及ALIM后,通过RT-PCR检测,发现TLR9mRNA在细菌或细菌组分刺激后的肺组织、外周血单核细胞上有表达,而在刺激后的上皮细胞A549中的无表达；在RT-PCR基础上进行流式细胞仪检测,发现TLR9在外周血单核细胞中经活性NTHi及细菌DNA激活后有表达增加,氯喹可抑制TLR9的表达,而肺泡上皮细胞A549经活性NTHi及细菌DNA刺激不表达TLR9蛋白；Western blot检测,ALIM肺组织经活性NTHi.细菌DNA及合成的ODN M362刺激后的TLR9有上调,MAPKp38p及ERK p44/42信号通路参与了TLR9的表达。 上述结果表明,气道及细胞对细菌株的不同应答跟细胞反应类型或菌种相关,并不跟细菌的数量完全相关。NTHi为传统意义上被认为是胞外菌,我们的实验表明NTHi不但能进入胞内,且不单纯居留在胞内以躲避抗菌素的杀菌作用,同时仍有一定水平的炎症因子的释放,产生炎症反应；细胞内NTHi刺激细胞表达IL-8及TNF-a的水平大概相当于NTHi刺激细胞总炎症介质水平的10%至30%。除活性NTHi外,热灭活或庆大霉素灭活的NTHi在刺激肺泡上皮细胞或单核细胞及肺组织后,仍可致炎症介质IL-8或TNF-a表达增加。细胞内细菌及细菌DNA、合成的ODN对机体TLR9介导的单核细胞抗NTHi炎症防御有一定的作用,且可受氯喹调控。通过RT-PCR检测TLR9mRNA在细菌或细菌组分刺激后的肺组织、外周血单核细胞上有表达,而在刺激后的上皮细胞A549中的无表达,流式细胞仪检测TLR9在外周血单核细胞中在活性NTHi及细菌DNA激活后有表达增加,氯喹可抑制TLR9的表达。肺泡上皮细胞A549经活性NTHi及细菌DNA刺激不表达TLR9蛋白,Western blot检测,ALIM肺组织经活性NTHi、细菌DNA及合成的ODN M362刺激后的TLR9有上调,MAPKp38p及ERK p44/42信号通路参与了活性NTH刺激后的TLR9蛋白的表达。 结论： 1. NTHi不但能进入胞内,且在胞内产生炎症反应；细胞内NTHi刺激细胞表达炎症介质的水平大概相当于NTHi刺激细胞总炎症介质水平的10%至30%。NTHi与呼吸道之间相互作用,NTHi可侵入并存留在上皮细胞及单核细胞内,刺激细胞释放炎症因子,保持呼吸道处于慢性炎症状态,NTHi的这种作用是COPD急性加重及慢性炎症的基础。 2.灭活的NTHi同活性细菌一样,可产生较强的炎症反应,而NTHi DNA和合成的ODN M362具有弱免疫活性。 3.外周血单核细胞及肺组织均表达TLR9mRNA及TLR9蛋白,但人上皮细胞A549不感知TLR9。TLR9作为一种细胞内受体,在上皮细胞中不表达,而在单核细胞中表达,且经刺激后TLR9表达上调。说明上皮细胞对细胞内细菌来说,无免疫细胞活性。 4. ALIM,可作为连接人体细胞和整体动物模型的桥梁,使我们可以在组织水平研究肺部感染性疾病发生发展的连续横断面变化。构建的急性肺组织NTHi感染模型,为我们研究细菌与宿主之前的相互作用提供了良好的平台。
[Abstract]:In higher vertebrates developed two protective immune system, natural immunity and acquired immunity. The natural immune system mainly depends on the perception of pathogen associated molecular patterns (pathogen-associated molecular, patterns, PAMPs) protein.TLRs (Toll-like receptors) pathogen recognition is a pattern recognition receptor of TLR, identify a series of bacteria, fungi and viruses unlike other products, TLR9 LTRs, in cells and identification of intracellular bacterial DNA. methylation
With nontypeable Haemophilus influenzae respiratory tract infection (non-typeable Haemophilus Influenza, NHTi) with correlation, especially the lungs and middle ear infection, with the incidence and mortality of.NTHi is a non capsular gram-negative bacillus multiforme, often in the majority of upper airway colonization in the body, or in the function of airway surface damage when the loss often cause opportunistic infections of.NTHi pathogens traditionally considered an extracellular bacterial non-invasive mucociliary clearance, but recent studies indicate that NTHi can be internalized into eukaryotic cells, invasion of epithelial cells and macrophages and survival in cells, so as to avoid antibiotic bactericidal activity.
We used antibiotic protection strategy, the cells incubated with NTHi 2H; through microscope observe NTHi into cells, NTHi can induce inflammatory reaction in extracellular and internalized into the cell, so the NTHi at the same time as facultative intracellular bacteria exist; at the same time, we construct acute pulmonary NTHi infection the model, by in situ hybridization in NTHi after ALIM4h stimulation in the alveolar cavity could be detected in the presence of NTHi.
Application of APA strategy to produce quantitative detection of NTHi stimulated human A549 cells and peripheral blood mononuclear cells and lung tissue after the inflammation factor IL-8 and / or TNF-a level, compared the total inflammatory cytokines and inflammatory cytokines production of intracellular NTHi and intracellular bacteria in the non dominant position of.ALIM cytokines stimulated by NTHi inflammation the release of cytokines. NTHi activity produced by comparison with inactivated bacteria had no significant differences in inflammation effect; bacterial components such as bacteria can induce DNA cells to produce inflammatory mediators, this effect could be endosomal maturation inhibitor chloroquine blocking.
The activity of NTHi, NTHi and DNA live bacteria killing stimulate epithelial A549 cells and peripheral blood mononuclear cells and ALIM, detected by RT-PCR, TLR9mRNA found in bacteria or bacterial components after the stimulation of lung tissue, peripheral blood mononuclear cells on the expression, but no expression in epithelial cells stimulated by A549 the flow cytometry; on the basis of RT-PCR, TLR9 in peripheral blood mononuclear cells by activated NTHi and bacterial DNA activation have increased expression of chloroquine inhibited TLR9 expression, and alveolar epithelial cells by A549 stimulation and NTHi activity of bacteria DNA TLR9 expression; Western blot detection. The activity of ALIM in lung tissue of NTHi. bacterial DNA and synthetic ODN M362 stimulated TLR9 increases, MAPKp38p and ERK p44/42 signaling pathway involved in the expression of TLR9.
The results showed that the cells of airway and bacterial strains in different cell types or strains in response to the reaction, not with the number of bacteria related to.NTHi is traditionally considered extracellular bacteria, our experiments show that NTHi not only can enter the cells, and not simply stay in intracellular to avoid the bactericidal effect of antibiotics at the same time, there is still a certain level of inflammatory factors release of inflammatory reaction; the level of NTHi cells stimulated the expression of IL-8 and TNF-a in vitro, roughly equivalent to the total cell NTHi stimulated inflammatory mediators levels of 10% to 30%. in addition to NTHi activity, heat inactivated or gentamicin inactivated NTHi in stimulating alveolar epithelial cells or mononuclear cells and the lung tissue, can still cause inflammation or increase the expression of TNF-a. IL-8 bacteria and bacterial DNA cells, the synthesis of ODN inflammatory defense has some effects on monocytes of body TLR9 mediated anti NTHi, and Can be detected by RT-PCR TLR9mRNA. The regulation of chloroquine in bacteria or bacterial components after the stimulation of lung tissue, peripheral blood mononuclear cells on expression in epithelial cells after A549 stimulation in expression, flow cytometry was used to detect the activation of TLR9 in active NTHi and bacterial DNA in peripheral blood mononuclear cells after the expression of increased expression of chloroquine inhibited TLR9. Alveolar epithelial cells A549 by stimulating activity of NTHi and bacterial expression of DNA TLR9 protein, Western blot detection of lung tissue ALIM activity by NTHi, bacterial DNA and synthetic M362 stimulated ODN TLR9 has raised the expression of MAPKp38p and ERK, p44/42 signaling pathway is involved in the activity NTH stimulated TLR9 protein.
Conclusion:
1. NTHi not only can enter the cells, and inflammatory reaction in the intracellular level of NTHi cells; stimulate the expression of inflammation cells is roughly equivalent to the stimulation of NTHi interaction between total cell inflammatory mediators levels of 10% to 30%.NTHi and NTHi can invade the respiratory tract, and retained in epithelial cells and mononuclear cells, stimulate the release of inflammatory cells factor, keeping respiratory tract in chronic inflammatory state, the effect of NTHi is the basis of acute exacerbation of COPD and chronic inflammation.
2. inactivated NTHi, like active bacteria, can produce strong inflammatory reactions, while NTHi DNA and synthetic ODN M362 have weak immunological activity.
TLR9mRNA and TLR9 protein was expressed in cells of lung tissue and 3. peripheral blood monocytes, but not A549 aware TLR9.TLR9 human epithelial cells as an intracellular receptor, not expressed in epithelial cells, while the expression in monocytes, and after stimulation with TLR9 upregulation. That epithelial cells of bacteria cells in no, the activity of immune cells.
4. ALIM, can be used as a bridge to connect human cells and animal models, so that we can change the occurrence and development of cross section in continuous lung disease level of tissue infection. Construction of lung tissue of acute NTHi infection model, before we study the bacteria and host interaction provides a good platform.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R392.1

【引证文献】

相关期刊论文 前1条

1 李海超;韩凌霞;;鸡抗病毒免疫相关的TLR信号途径研究进展[J];动物医学进展;2013年08期

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