呼吸道上皮细胞与致病菌相互作用--上皮细胞内吞细菌实验研究

目的：呼吸道上皮细胞在防御这类机会致病菌感染时发挥了重要的作用。本研究旨在探讨上皮细胞能否清除胞内的绿脓杆菌，胞内模式识别受体Nods蛋白家族是否参与胞内杀菌，防御素是否能通过促经克雷伯杆菌粘附到上皮细胞而被上皮细胞内在化而清除。方法：首先用绿脓杆菌活菌刺激支气管原代上皮细胞及A549细胞，活菌细胞共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续孵育4小时，24小时后，用TritonX-100溶解细胞，采用平板菌落计数法计数胞内活菌数。绿脓杆菌与细胞按不同比例共孵育两小时后庆大霉素杀死未进入胞内的细菌，继续培养24小时后收集细胞培养上清，酶联免疫吸附法（ELISA）检测IL-8的表达量。为进一步研究Nods蛋白是否在胞内杀菌及活菌在胞内引起IL-8分泌中发挥作用，我们用超声处理的绿脓杆菌菌体成分刺激使细胞膜通透性增加的温和去污剂digitonin处理和未使用digitonin处理A549细胞，ELISA检测细胞IL-8表达水平。胞内模式识别受体Nod1，Nod2可以识别菌体成分，用RT-PCR检测肺上皮细胞Nodl，Nod2的表达。其次，     

肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL-8的研究

目的 :探讨肺炎克雷伯杆菌 (Klebsiellapneumoniae ,Kp)分泌因子及活菌诱导肺上皮细胞株表达和分泌IL 8的状况。方法 :用临床分离株Kp0 3 1 1 6、Kp0 3 1 83的细菌培养上清或活菌刺激肺上皮细胞株A5 49和SPC A 1 ,酶联免疫吸附实验 (ELISA)检测细胞IL 8表达量。结果 :①两株Kp培养上清分别刺激A5 49或SPC A 1后 ,IL 8表达量均有显著增高 (P <0 .0 1 ) ,且随上清刺激浓度增加而上升。②两株Kp及DH5 (活菌分别与SPC A 1共孵育 2h ,用庆大霉素杀死胞外菌 ,继续孵育 2 4h后两株Kp诱导细胞IL 8表达量均显著高于DH5α(P <0 .0 1 ) ,且随细菌数 /细胞数比例增大而上升。结论 :Kp分泌因子及活菌都能够诱导肺上皮细胞IL 8表达 ,而活菌上调IL 8的效应较培育上清分泌因子更显著 ,提示肺上皮细胞在Kp感染刺激的肺部炎症反应中起重要作用。     

MyD88缺去突变基因转染降低病原菌感染的人呼吸道上皮细胞IL-8的分泌

白细胞介素-8（1L-8）是呼吸道炎症反应的重要介质。本实验通过构建突变MyD88真核表达质粒（MyD88 DN），转染人呼吸道上皮细胞株A549及SPC-A-1，探讨其对病原菌感染上皮细胞IL-8表达的影响。结果显示：MyD88 DN转染可降低结核杆菌、绿脓杆菌培养上清诱导的IL-8释放；对肺炎克雷伯杆菌和绿脓杆菌活菌侵袭细胞所刺激的IL-8分泌也有明显的阻断作用。提示突变MyD88能够阻断细菌感染引起的呼吸道上皮细胞IL-8表达，可能成为呼吸道严重炎症反应基因治疗的新靶基因。     

TNF及铜绿假单胞菌刺激呼吸道上皮细胞表达NOD2蛋白

目的：通过对TNF及铜绿假单胞菌（PAO1）刺激肺上皮细胞株（A549）表达胞浆蛋白NOD2的研究,进一步了解NOD2在机体天然免疫病原体识别过程中的作用。方法：培养呼吸道肺癌上皮细胞株A549,以TNF、铜绿假单胞菌PAO1刺激细胞,并以未刺激组做对照,通过半定量RT-PCR技术观察胞浆蛋白NOD2的表达变化。结果：与未刺激组比较,TNF、铜绿假单胞菌PAOI都能够上调A549细胞NOD2蛋白的表达。结论：TNFα与铜绿假单胞菌PAO1刺激能增强A549细胞胞装蛋白NOD2的表达,提示细胞内受体NOD2在呼吸道天然免疫病原体识别过程中可能起重要作用。     

腺病毒介导突变Rip2基因对膀胱上皮细胞清除克雷伯杆菌的影响

目的探讨受体相互作用蛋白2（Rip2）在克雷伯杆菌所致的尿路感染中的作用。方法使用含有突变Rip2基因（mRip2）、绿色荧光蛋白（GFP）基因的复制缺陷腺病毒（Ad-mRip2-GFP）感染人膀胱上皮癌细胞T24细胞，表达GFP为转染成功标志，观察转染率，同时感染含有GFP基因腺病毒做对照，利用克雷伯杆菌临床分离株Kp5与细胞37℃孵育4、24、48小时，用平板培养菌落计数活菌数。结臬刺激后，在感染Ad-mRip2-GFP的T24细胞清除胞内Kp5能力下降，与Ad-GFP组比较P〈0．01。结论Rip2可以阻断膀胱上皮细胞的清除克雷伯杆菌作用，提示Rip2在膀胱上皮细胞的天然免疫中可能起重要作用。     

HNP对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响

目的：探讨呼吸道抗感染防御机制，观察人中性粒细胞α-防御素（HNP）对肺炎克雷伯杆菌粘附呼吸道上皮细胞的影响。方法：从人中性粒细胞分离纯化HNP。A549细胞与HNP（20μg·ml^-1）及两株肺炎克雷伯杆菌临床分离株共同孵育4小时，用平板培养茵落计数法测定与细胞粘附的活茵数。结果：在HNP存在的情况下，Kp03.33株细菌对肺上皮细胞的粘附提高了12倍（P〈0.01），Kp03116株细菌对上皮细胞的粘附提高了5倍（p〈0.01）结论：HNP显著增强肺炎克雷伯杆菌粘附于呼吸道上皮细胞，可能有利于呼吸道清除细菌。     

《四川生理科学杂志》2004年目录索引

目　　次论　　著1995～ 2 0 0 2年阴沟肠杆菌临床分离株的耐药性分析马越　李景云　张新妹等 (1)………………………………人子宫内膜粘液抗菌多肽的分离纯化王莉莉　潘小玲　黄宁等 (5 )………………………………………………肺炎克雷伯杆菌分泌因子及活菌诱导人肺上皮细胞表达分泌IL 8的研究陈襄文　王芳　周敏燕等 (7)……………结核分支杆菌培养上清诱导呼吸道上皮细胞IL 8表达王芳　冯艳　陈襄文等 (10 )………………………………三七皂甙R1诱导HL 6 0细胞凋亡的初步研究王国俊　周黎明　王莉等 (14 )………………………………………     

肺炎克雷伯杆菌荚膜多糖经EGFR途径诱导支气管上皮细胞分泌细胞因子

目的:探讨表皮生长因子受体(EGFR)在肺炎克雷伯杆菌(KP)荚膜多糖(CPS)诱导人正常支气管上皮BEAS-2B细胞分泌炎性细胞因子中的作用。方法:体外培养KP并提取CPS,用不同浓度的CPS刺激BEAS-2B细胞,通过ELISA检测细胞上清中肿瘤坏死因子α(TNF-α)和白细胞介素8(IL-8)的水平,并于刺激后不同时点通过Western blot检测EGFR的磷酸化水平;EGFR抑制剂AG1478预处理BEAS-2B细胞后,Western blot检测ERK磷酸化水平,间接免疫荧光染色检测p65的核转位,并观察细胞上清中TNF-α和IL-8的变化情况;最后经ERK抑制剂PD98059和NF-κB抑制剂PDTC分别预处理后用CPS刺激细胞,ELISA检测细胞上清中TNF-α和IL-8的水平。结果:10 mg/L CPS刺激BEAS-2B细胞12 h能够显著诱导其分泌TNF-α和IL-8;Western blot和间接免疫荧光染色检测结果显示,CPS刺激可显著诱导BEAS-2B细胞中EGFR和ERK的磷酸化及p65的核转位。经EGFR抑制剂AG1478预处理细胞后,ERK的磷酸化水平显著降低,NF-κB的核转位减少;而在EGFR、ERK及NF-κB抑制剂预处理的细胞的上清中,TNF-α和IL-8分泌水平均明显降低(P<0.05)。结论:肺炎克雷伯杆菌荚膜多糖能够通过EGFR激活ERK和NF-κB信号通路,进而诱导人正常支气管上皮细胞分泌炎性细胞因子TNF-α和IL-8,提示EGFR可能是KP感染引起炎症反应的关键因子。     

TNF-α增强膀胱上皮细胞清除细胞内肺炎克雷伯杆菌

研究TNF-α对膀胱上皮细胞内肺炎克雷伯杆菌生存的影响。方法：以肺炎克雷伯杆菌侵入体外培养的人膀胱上皮细胞（T24细胞）为模型,观察在TNF-α等细胞因子处理条件下,不同时间点细胞内细菌数量变化。结果：单独使用TNF-α使T24细胞内的肺炎克雷伯杆菌K5株细菌数量明显减少,联合使用TNF-α与IFN-γ使胞内活菌数量更显著的减少。而IL-1β对细胞内活菌数量无明显影响。过氧化氢酶可以有效抑制TNF-α与IFN-γ刺激的＂1＂24细胞抗菌作用,而一氧化氮合酶抑制剂L-NAME无抑制作用。结论：TNF-α能够增强膀胱上皮细胞对抗细胞内肺炎克雷伯杆菌,抗菌机制与细胞产生活性氧（ROS）有关。     

肺炎克雷伯杆菌在T24细胞内存活的动态观察

目的：了解肺炎克雷伯杆菌与膀胱上皮细胞的相互关系，观察肺炎克雷伯杆菌在人膀胱上皮细胞抹T24中生存的动态变化。方法：采用肺炎克雷伯杆菌临床分离抹03138侵袭T24细胞，并用庆大霉素杀死细胞外的细菌，分别于细菌进入细胞后的4、24、48及72h裂解细胞，释放出细胞内的活细菌，用平板菌落计数法计数胞内活菌数。结果：T24细胞内的肺炎克雷伯杆菌03138抹在实验48h内有一定生长，试验72h细胞内活菌数量明显减少。加入细胞因子（TNF-αd和INF-γ）可以促进上皮细胞清除胞内细菌。结论：膀胱上皮细胞清除进入细胞内的肺炎克雷伯杆菌，可能是泌尿道天然免疫的一种防御机制，而细胞因子可以调控上皮细胞的抗菌作用。     

Induction of proinflammatory cytokines in human lung epithelial cells during Chlamydia pneumoniae infection

Chlamydia pneumoniae is an obligate intracellular human pathogen that causes acute respiratory diseases such as pneumonia and bronchitis. Previous studies have established that C. pneumoniae can induce cytokines in mouse and/or human cells, but little information is available on the cytokine response of respiratory epithelial cells, a first line of infection. In this study, heparin treatment of C. pneumoniae significantly reduced its ability to induce interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) mRNA in human lung carcinoma cells, indicating that cytadherence is an important early stimulus for induction of proinflammatory mediators. Although the IL-8, gamma interferon, and TNF-alpha message was consistently induced by infection of A549 cells not treated with heparin, only an elevation of IL-8 protein was detected in A549 supernatants. A549 IL-beta and IL-6 mRNA and supernatant protein profiles were not significantly changed by infection. Heat or UV inactivation of C. pneumoniae only partially reduced the cytokine response, and inhibition of C. pneumoniae protein or DNA synthesis did not affect its ability to induce cytokine gene expression. To prevent stress-induced cytokine release by the A549 cells, centrifugation was not utilized for infection experiments. These experiments establish the importance of cytadherence in cytokine release by cells of respiratory epithelial origin and suggest that further work in the area of cytokine mediators is warranted to gain valuable pathogenic and therapeutic insights.     

铜绿假单胞菌活菌对呼吸道上皮细胞表达IL-8的影响

目的:探讨铜绿假单胞菌活菌与人呼吸道上皮细胞的相互关系,细菌对呼吸道上皮炎症反应的影响。方法:采用PAO1及ATCC 27853两株铜绿假单胞菌,在体外与培养的呼吸道上皮细胞株A549及无血清培养的人支气管上皮原代细胞相互作用,收集细胞培养上清,ELISA检测上清IL-8浓度。结果:两株绿脓杆菌均能诱导呼吸道上皮细胞IL-8分泌增加,在细菌刺激下,A549细胞IL-8分泌比对照高出5倍(P<0.05),原代上皮细胞IL-8分泌比对照高出8倍(P<0.05)。结论:铜绿假单胞菌呼吸道感染的过程中,细菌与上皮细胞的直接作用可能是呼吸道炎症反应的重要原因。铜绿假单胞菌刺激上皮细胞炎症的分子机制和信号传导值得进一步探讨。     

Caspase-3基因沉默对肺炎链球菌感染的肺泡上皮细胞凋亡的影响

目的:通过siRNA抑制caspase-3基因的表达探讨肺炎链球菌对肺泡上皮细胞凋亡的影响及凋亡基因caspase-3对凋亡的调节作用,寻找治疗肺炎链球菌肺炎的新方法。方法:体外培养肺泡上皮细胞A549,用肺炎链球菌R6作用于A549细胞,使用siRNA技术抑制caspase-3表达,RT-PCT法检测caspase-3转录强度,化学荧光测定法检测caspase-3蛋白含量,ELISA法检测细胞上清中IL-6和IL-10浓度,TUNEL法检测细胞凋亡。结果:肺炎链球菌能诱导A549细胞凋亡、导致caspase-3的表达和IL-6的浓度升高、IL-10的浓度降低;使用siRNA抑制caspase-3表达后,caspase-3的表达降低,A549细胞的凋亡率降低,而IL-6和IL-10的浓度并无明显变化。结论:Caspase-3在肺炎链球菌引起的肺泡上皮细胞的凋亡中占据了重要的地位;运用RNA干扰技术抑制caspase-3的表达能降低肺泡上皮细胞的凋亡率,这可能对肺炎链球菌肺炎的治疗有积极意义。     

Regulation of proinflammatory cytokines in human lung epithelial cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans. It has also been associated with chronic conditions, such as arthritis, and extrapulmonary complications, such as encephalitis. Although the interaction of mycoplasmas with respiratory epithelial cells is a critical early phase of pathogenesis, little is known about the cascade of events initiated by infection of respiratory epithelial cells by mycoplasmas. Previous studies have shown that M. pneumoniae can induce proinflammatory cytokines in several different study systems including cultured murine and human monocytes. In this study, we demonstrate that M. pneumoniae infection also induces proinflammatory cytokine expression in A549 human lung carcinoma cells. Infection of A549 cells resulted in increased levels of interleukin-8 (IL-8) and tumor necrosis factor alpha mRNA, and both proteins were secreted into culture medium. IL-1 beta mRNA also increased after infection and IL-1 beta protein was synthesized, but it remained intracellular. In contrast, levels of IL-6 and gamma interferon mRNA and protein remained unchanged or undetectable. Using protease digestion and antibody blocking methods, we found that M. pneumoniae cytoadherence is important for the induction of cytokines. On the other hand, while M. pneumoniae protein synthesis and DNA synthesis do not appear to be prerequisites for the induction of cytokine gene expression, A549 cellular de novo protein synthesis is responsible for the increased cytokine protein levels. These results suggest a novel role for lung epithelial cells in the pathogenesis of M. pneumoniae infection and provide a better understanding of M. pneumoniae pathology at the cellular level.     

Endotoxin potency in the A549 lung epithelial cell bioassay and the limulus amebocyte lysate assay.

The purpose of this study was to elucidate to what extent the potency of endotoxins measured by the limulus amebocyte lysate (LAL) assay is reflected in the potency in an in vitro assay based on release of interleukin-8 (IL-8) from a lung epithelial cell line, A549. Lipopolysaccharides (LPS) from Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enteritidis and detoxified LPS from E. coli were applied in serial dilutions in the LAL assay and in the A549 bioassay. Also 19 organic dust samples from waste recycling plants were tested. The A549 cells were incubated for 24 h with LPS or dust, and the IL-8 secretion was determined by ELISA. The method for evaluation of the LAL assay showed linearity for the four endotoxins. Using the slope as a measure of the potency factor (PF), LPS from E. coli and S. enteritidis was about four times more potent than that for LPS from K. pneumoniae and P. aeruginosa. In the A549 bioassay each of the different types of endotoxin had characteristic and very different dose-response curves. The potency of the LPS, in the A549 bioassay, ranked as follows K. pneumoniae > P. aeruginosa > E. coli > or = S. enteritidis. The content of endotoxin in the dust samples did not correlate with their potency in the A549 bioassay. The present study indicates a poor correlation between the potency of endotoxin in the LAL assay compared with the A549 bioassay. The lack of correlation when organic dust samples are tested may reflect the fact that these samples contain biological active compounds, which are non-reactive in the LAL-assay but stimulate IL-8 secretion from epithelial cells.     

结核分支杆菌培养上清诱导呼吸道上皮细胞IL-8表达

目的：观察呼吸道上皮细胞对结核杆菌产物产生的炎症反应，探讨炎症反应信号传递的机制。方法：制备结核分枝杆菌强毒力H37RV株培养上清，用培养上清刺激肺上皮细胞株SPC-A-1和A549，酶联免疫吸附实验检测细胞IL-8表达量。用突变MyD88表达重组质粒转染SPC-A-1细胞，观察MyD88与结核杆菌培养上清诱导上皮细胞炎症反应信号传递的关系。结果：结核分枝杆菌强毒力H37RV株培养上清明显增加SPC-A-1和A549细胞IL-8的表达。用突变MyD88表达重组质粒pcDNA3．1-MyD88转染SPC-A-1。结果显著抑制了结核杆菌培养上清对SPC-A-l细胞的炎症刺激作用。结论：结核杆菌培养上清能够诱导呼吸道上皮细胞IL-8表达，，Toll／IL-1受体信号通路及MyD88分子与细胞炎症反应的信号传递相关，提示在肺结核病发病过程中。结核杆菌的代谢产物可能刺激肺上皮细胞产生炎症细胞因子，参与肺结核炎症病变的形成。     

Interleukin-1beta responses to Mycoplasma pneumoniae infection are cell-type specific

Interleukin-1beta (IL-1beta) is a major proinflammatory cytokine that is involved in many important cellular functions such as proliferation, differentiation, and activation of different cell types. Its mature form is released from the cells in response to various bacterial and viral infections, and it plays a significant role in host defense. Mycoplasma pneumoniae is a small bacterium without a cell wall that causes tracheobronchitis and atypical pneumonia in humans following attachment to respiratory epithelium, as well as extrapulmonary infections. Very little is known about the role of cytokines in pathogenesis or the response of target cells to M.pneumoniae attachment. The purpose of this study was to investigate the ability of M. pneumoniae to induce IL-1beta in human lung epithelial carcinoma A549 and in human monocytic U937 cell lines. Following M. pneumoniae infection, both IL-1beta mRNA and protein were induced in A549 cells vs. no induction in uninfected cells; however, the protein remained inside the A549 cells. Similarly, M. pneumoniae infection strongly increased mRNA and extracellular protein levels in U937 cells, which unlike A549 cells did exhibit baseline constitutive levels. De novo IL-1beta protein expression was verified by cycloheximide studies. M. pneumoniae infection did not affect constitutive caspase-1 mRNA or protein levels in either cell line. Reduced caspase-1 activity in A549 cell lysates suggests the presence of an endogenous caspase-1 inhibitory component in the A549 cells. These collective data confirm previous studies that show that M. pneumoniae is a potent inducer of cytokines following adherence to host target cells, and establish that IL-1beta release in response to M. pneumoniae infection is cell-type specific, thus emphasizing the importance of carefully considering multiple cell types in M. pneumoniae pathogenesis studies involving both immune cells and cytokine release patterns.     

C3 promotes clearance of Klebsiella pneumoniae by A549 epithelial cells

The airway epithelium represents a primary site for contact between microbes and their hosts. To assess the role of complement in this event, we studied the interaction between the A549 cell line derived from human alveolar epithelial cells and a major nosocomial pathogen, Klebsiella pneumoniae, in the presence of serum. In vitro, we found that C3 opsonization of poorly encapsulated K. pneumoniae clinical isolates and an unencapsulated mutant enhanced dramatically bacterial internalization by A549 epithelial cells compared to highly encapsulated clinical isolates. Local complement components (either present in the human bronchoalveolar lavage or produced by A549 epithelial cells) were sufficient to opsonize K. pneumoniae. CD46 could competitively inhibit the internalization of K. pneumoniae by the epithelial cells, suggesting that CD46 is a receptor for the binding of complement-opsonized K. pneumoniae to these cells. We observed that poorly encapsulated strains appeared into the alveolar epithelial cells in vivo but that (by contrast) they were completely avirulent in a mouse model of pneumonia compared to the highly encapsulated strains. Our results show that bacterial opsonization by complement enhances the internalization of the avirulent microorganisms by nonphagocytic cells such as A549 epithelial cells and allows an efficient innate defense.     

视黄酸对细胞因子诱导的人肺泡Ⅱ型上皮细胞分泌C3及B因子的影响

目的:探讨视黄酸(RA)对细胞因子诱导的人肺泡Ⅱ型上皮细胞A549分泌C3及B因子的影响。方法:用ELISA检测TNFα和IL1β诱导的A549细胞培养上清中C3及B因子的水平。用RTPCR分析C3及B因子mRNA的表达。结果:TNFα和IL1β诱导A549细胞分泌C3及B因子具有时间和剂量依赖性。IL6诱导A549细胞分泌C3和B因子的水平是未处理组的4.7、1.4倍,IFNγ诱导A549细胞分泌C3和B因子的水平是未处理组的2.1、1.7倍。RA本身对A549细胞分泌C3及B因子没有影响,但可显著增强TNFα和IL1β诱导的A549细胞分泌C3和B因子及其mRNA的表达,以及IL6和IFNγ诱导的B因子合成。结论:RA可上调TNFα、IL1β、IL6和IFNγ诱导的A549细胞分泌C3及B因子,调节肺局部组织的免疫防御反应,为临床上应用RA和细胞因子防治肺部疾病提供了理论依据。     

Neutrophil defensins stimulate the release of cytokines by airway epithelial cells: modulation by dexamethasone

OBJECTIVE AND DESIGN: Neutrophils may contribute to recruiting other cells to sites of inflammation by generating chemotactic signals themselves, or by stimulating other cell types to release chemoattractants such as interleukin-8 (IL-8). Recently, we demonstrated that neutrophil-derived alpha-defensins are able to increase IL-8 expression in airway epithelial cells. In addition, it has previously been reported that neutrophil elastase-induced IL-8 synthesis was insensitive to inhibition by the glucocorticoid dexamethasone. The aim of the present study was to investigate the effect of defensins on the expression of various cytokines in cultured airway epithelial cells and to examine the effect of dexamethasone on defensin-induced cytokine synthesis in these cells. METHODS: Cultures of A549 cells and primary bronchial epithelial cells (PBEC) were stimulated with defensins either alone or in the presence of dexamethasone. Supernatants were analyzed for IL-8, ENA-78, IL-6, MCP-1 and GM-CSF by ELISA. In addition, IL-8 and ENA-78 mRNA was detected by Northern blot analysis. RESULTS: Defensins increased IL-8 expression, ENA-78, MCP-1 and GM-CSF release from A549 cells, whereas in PBEC only IL-8 and IL-6 were increased. Pre-treatment with dexamethasone significantly reduced defensin-induced IL-6, IL-8 and ENA-78 synthesis in airway epithelial cells. In addition, dexamethasone also reduced the neutrophil chemotactic activity in supernatants of these cells. CONCLUSIONS: The results from the present study indicate that defensins differentially induce cytokine secretion by A549 cells and PBEC. Glucocorticoids may interfere with the defensin-induced inflammatory process by reducing defensin-induced cytokine secretion in lung epithelial cells.