本文關(guān)鍵詞：不可分型流感嗜血桿菌在呼吸道感染中的免疫應(yīng)答對細胞內(nèi)受體TLR9在感染中的表達研究　出處：《浙江大學(xué)》2012年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 流感嗜血桿菌 呼吸道感染 細胞內(nèi)受體 TLR9

【摘要】：高級脊椎動物發(fā)展了兩套保護性免疫系統(tǒng),天然免疫和獲得性免疫。天然免疫系統(tǒng)主要依賴于通過感知病原體相關(guān)分子模式(pathogen-associated molecular patterns, PAMPs)的蛋白質(zhì)識別病原體。TLRs (toll-like receptors)是研究較多的模式識別受體,TLR識別一系列的細菌、真菌及病毒產(chǎn)物,TLR9與其他LTRs不同,存在于細胞內(nèi),識別細胞內(nèi)非甲基化細菌DNA。 呼吸道感染與不可分型流感嗜血桿菌(non-typeable Haemophilus Influenza, NHTi)具有相關(guān)性,特別是肺部及中耳細菌感染,具有一定的發(fā)病率及死亡率。NTHi是一種無莢膜的革蘭氏陰性多形桿菌,常定植于大多數(shù)人的上氣道,在機體氣道表面受損或在粘液纖毛清除功能散失時常常引起機會性感染的病原體。NTHi傳統(tǒng)意義上被認為是一種非侵入性的細胞外細菌,但近期研究表明,NTHi可內(nèi)化至真核細胞內(nèi),侵入上皮細胞及巨噬細胞并在細胞內(nèi)生存,從而躲避抗菌素的殺菌活性。 我們應(yīng)用抗菌素保護策略,將細胞同NTHi共孵育2h后；通過高清顯微鏡直觀的觀察NTHi進入到細胞內(nèi),NTHi可在胞外產(chǎn)生炎癥反應(yīng)的同時,內(nèi)化入細胞內(nèi),故NTHi同時作為兼性胞內(nèi)菌存在；同時,我們構(gòu)建了急性肺部NTHi感染模型,應(yīng)用原位雜交技術(shù),在NTHi刺激ALIM4h后,在肺泡腔內(nèi)可檢測到NTHi的存在。 應(yīng)用APA策略,定量檢測到NTHi刺激后人上皮細胞A549及外周血單核細胞及肺組織后產(chǎn)生的炎癥因子IL-8和／或TNF-a水平,總炎癥因子與胞內(nèi)NTHi產(chǎn)生的炎癥因子比較,胞內(nèi)菌炎癥因子處于非優(yōu)勢地位。ALIM經(jīng)NTHi刺激后有炎癥因子的釋放。活性NTHi所產(chǎn)生炎癥效應(yīng)同滅活菌比較無顯著差異；細菌成分如細菌DNA,可誘導(dǎo)細胞產(chǎn)生炎癥介質(zhì),此作用可被核內(nèi)體成熟抑制劑氯喹阻斷。 將活性NTHi、滅活的NTHi及細菌DNA刺激上皮細胞A549及外周血單核細胞及ALIM后,通過RT-PCR檢測,發(fā)現(xiàn)TLR9mRNA在細菌或細菌組分刺激后的肺組織、外周血單核細胞上有表達,而在刺激后的上皮細胞A549中的無表達；在RT-PCR基礎(chǔ)上進行流式細胞儀檢測,發(fā)現(xiàn)TLR9在外周血單核細胞中經(jīng)活性NTHi及細菌DNA激活后有表達增加,氯喹可抑制TLR9的表達,而肺泡上皮細胞A549經(jīng)活性NTHi及細菌DNA刺激不表達TLR9蛋白；Western blot檢測,ALIM肺組織經(jīng)活性NTHi.細菌DNA及合成的ODN M362刺激后的TLR9有上調(diào),MAPKp38p及ERK p44/42信號通路參與了TLR9的表達。 上述結(jié)果表明,氣道及細胞對細菌株的不同應(yīng)答跟細胞反應(yīng)類型或菌種相關(guān),并不跟細菌的數(shù)量完全相關(guān)。NTHi為傳統(tǒng)意義上被認為是胞外菌,我們的實驗表明NTHi不但能進入胞內(nèi),且不單純居留在胞內(nèi)以躲避抗菌素的殺菌作用,同時仍有一定水平的炎癥因子的釋放,產(chǎn)生炎癥反應(yīng)；細胞內(nèi)NTHi刺激細胞表達IL-8及TNF-a的水平大概相當(dāng)于NTHi刺激細胞總炎癥介質(zhì)水平的10%至30%。除活性NTHi外,熱滅活或慶大霉素滅活的NTHi在刺激肺泡上皮細胞或單核細胞及肺組織后,仍可致炎癥介質(zhì)IL-8或TNF-a表達增加。細胞內(nèi)細菌及細菌DNA、合成的ODN對機體TLR9介導(dǎo)的單核細胞抗NTHi炎癥防御有一定的作用,且可受氯喹調(diào)控。通過RT-PCR檢測TLR9mRNA在細菌或細菌組分刺激后的肺組織、外周血單核細胞上有表達,而在刺激后的上皮細胞A549中的無表達,流式細胞儀檢測TLR9在外周血單核細胞中在活性NTHi及細菌DNA激活后有表達增加,氯喹可抑制TLR9的表達。肺泡上皮細胞A549經(jīng)活性NTHi及細菌DNA刺激不表達TLR9蛋白,Western blot檢測,ALIM肺組織經(jīng)活性NTHi、細菌DNA及合成的ODN M362刺激后的TLR9有上調(diào),MAPKp38p及ERK p44/42信號通路參與了活性NTH刺激后的TLR9蛋白的表達。 結(jié)論： 1. NTHi不但能進入胞內(nèi),且在胞內(nèi)產(chǎn)生炎癥反應(yīng)；細胞內(nèi)NTHi刺激細胞表達炎癥介質(zhì)的水平大概相當(dāng)于NTHi刺激細胞總炎癥介質(zhì)水平的10%至30%。NTHi與呼吸道之間相互作用,NTHi可侵入并存留在上皮細胞及單核細胞內(nèi),刺激細胞釋放炎癥因子,保持呼吸道處于慢性炎癥狀態(tài),NTHi的這種作用是COPD急性加重及慢性炎癥的基礎(chǔ)。 2.滅活的NTHi同活性細菌一樣,可產(chǎn)生較強的炎癥反應(yīng),而NTHi DNA和合成的ODN M362具有弱免疫活性。 3.外周血單核細胞及肺組織均表達TLR9mRNA及TLR9蛋白,但人上皮細胞A549不感知TLR9。TLR9作為一種細胞內(nèi)受體,在上皮細胞中不表達,而在單核細胞中表達,且經(jīng)刺激后TLR9表達上調(diào)。說明上皮細胞對細胞內(nèi)細菌來說,無免疫細胞活性。 4. ALIM,可作為連接人體細胞和整體動物模型的橋梁,使我們可以在組織水平研究肺部感染性疾病發(fā)生發(fā)展的連續(xù)橫斷面變化。構(gòu)建的急性肺組織NTHi感染模型,為我們研究細菌與宿主之前的相互作用提供了良好的平臺。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R392.1

【引證文獻】

相關(guān)期刊論文 前1條

1 李海超;韓凌霞;;雞抗病毒免疫相關(guān)的TLR信號途徑研究進展[J];動物醫(yī)學(xué)進展;2013年08期

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本文編號：1386817