【作者】 潘明鸣；

【导师】 孙铁英； 方保民； 柯会星；

【作者基本信息】 中国协和医科大学 ， 内科学， 2008， 博士

【摘要】 背景和目的Toll样受体（Toll like receptors,TLRs）可特异性识别多种病原成份,启动固有免疫反应,并进一步诱导适应性免疫应答。呼吸道感染及环境暴露因素对慢性阻塞性肺疾病（COPD）的发生、发展有重要影响。作为固有免疫的重要组成部分,TLRs是连接环境暴露与适应性免疫的重要桥梁,其在COPD发病机制中发挥的作用开始受到广泛关注。现有研究结果提示TLRs参与COPD的病理过程和免疫应答,但对TLRs在COPD患者体内实际表达情况和作用的研究很少。本课题在稳定期COPD患者中,以健康不吸烟者、肺功能正常吸烟者、慢性持续期支气管哮喘（简称哮喘）为对照,探讨TLRs在COPD发生发展及炎症特点中的作用;应用A549肺上皮肿瘤细胞株,探讨Th1相关细胞因子及糖皮质激素治疗对呼吸道上皮TLRs表达的调节,从而进一步明确TLRs在COPD发病机制中发挥的作用。研究对象、方法临床研究部分入选健康不吸烟者25例,肺功能正常吸烟者21例,稳定期COPD患者30例,慢性持续期哮喘患者30例。其中稳定期COPD患者均为吸烟者。应用流式细胞仪全血免疫荧光直标法检测所有受试者外周静脉血CD14+单核细胞及中性粒细胞的TLR2、TLR4表达。所有受试者均检测肺功能,COPD患行查痰培养、血清C反应蛋白（CRP）,应用酶连免疫吸附法（ELISA）测定COPD和哮喘患者血清TNF-α、IL-8浓度。实验室研究部分以A549人肺上皮肿瘤细胞株为实验对象,分别单独应用Th1相关细胞因子IFN-γ及TNF-α、地塞米松或联合应用地塞米松及上述细胞因子处理细胞,采用实时定量聚合酶链式反应（Real time PCR）法检测A549细胞TLR1、TLR2、TLR3、TLR4、TLR6表达的变化情况。研究结果临床研究部分1.CD14+单核细胞TLR2表达强度及阳性细胞率显著高于中性粒细胞（P＜0.001）。CD14+单核细胞TLR2表达与TLR4表达成正相关（P＜0.001）。CD14+单核细胞与中性粒细胞间TLR2、TLR4表达成正相关（P＜0.05）。2.COPD患者有24例留取合格痰标本,其中16例细菌负荷量超过10⁵CFU/ml,以革兰氏阴性杆菌为主;CRP均处于正常水平。3.健康不吸烟者、肺功能正常吸烟者及稳定期COPD患者3组比较:a.外周血CD14+单核细胞TLR2和TLR4的表达在肺功能正常吸烟者中明显下调（P＜0.05）,在稳定期COPD患者中进一步显著下降（P＜0.05）。b.外周血中性粒细胞TLR2表达在肺功能正常吸烟者中明显下调（P＜0.05）,在稳定期COPD患者中有进一步下降趋势（P＞0.05）。中性粒细胞TLR4表达情况3组间无明显差异（P＞0.05）。c.CD14+单核细胞TLR2和TLR4的表达强度,中性粒细胞TLR2的表达强度与FEV₁%预计值、FEV₁/FVC比值成显著正相关（P＜0.001）。4.慢性持续期哮喘患者CD14+单核细胞中TLR2及TLR4阳性细胞率明显高于健康对照组（P＜0.01）,中性粒细胞TLR2、TLR4的表达低于健康对照（P＜0.01）。CD14+单核细胞TLR2和TLR4的表达强度与哮喘患者血清IgE浓度有相关的趋势。5.COPD患者与哮喘患者的比较:a.COPD患者外周血中性粒细胞TLR2、TLR4表达明显高于哮喘患者（P＜0.05）b.COPD患者CD14+单核细胞TLR2、TLR4表达强度及阳性细胞率显著低于哮喘患者（P＜0.001）。c.COPD患者血清IL-8浓度水平显著高于哮喘患者,血清TNF-α浓度水平则低于哮喘患者（P＜0.01）。d.外周血中性粒细胞TLR2及TLR4表达强度与血清IL-8浓度成正相关（P＜0.01）,单核细胞TLR2及TLR4阳性细胞率与血清TNF-α浓度成正相关（P＜0.01）。实验室研究部分1.IFN-γ使A549细胞TLR3表达明显上调,达对照组的3.78倍（P＜0.05）。2.TNF-α使A549细胞TLR2、TLR6出现轻度上调（P＜0.05）,分别达到对照的1.62倍和1.22倍。3.地塞米松使A549细胞TLR2、TLR4表达明显上调,分别达对照的3.27及3.43倍（P＜0.05）。4.地塞米松与IFN-γ共同作用A549细胞时,TLR2表达显著上调,达对照的6.25倍,地塞米松与TNF-α联合作用时TLR2表达增加至对照6.09倍（P＜0.05）。5.TNF-α与IFN-γ联合刺激A549细胞时,TLR2上调达对照的3.66倍,而地塞米松与TNF-α、IFN-γ三者共同作用A549细胞时,TLR2的表达出现十分显著的上调,达对照的29.81倍（P＜0.05）。结论1.健康人群单核细胞TLR2、TLR4表达显著高于中性粒细胞。2.吸烟抑制单核细胞和中性粒细胞TLRs表达。3.COPD患者单核细胞和中性粒细胞TLRs表达受到抑制,TLRs表达下调与病情进展有关。4.COPD患者TLRs的表达状态与其炎症反应特点有关。5.Th1细胞因子及糖皮质激素上调呼吸道上皮细胞TLRs表达,二者对呼吸道上皮细胞TLR2表达的上调具有协同作用,提示吸入激素治疗可能增强COPD患者气道上皮防御功能。更多还原

【Abstract】 BackgroundToll-like receptors (TLRs) are involved in recognition of a broad range of pathogens. Their activation triggers the innate immune response, and is crucial to the successful induction of adaptive immunity. Respiratory infections and environmental stimuli influence the risk of developing COPD and the disease progression. As the key receptors of the innate immune system, TLRs are the important link between environmental exposure and regulation of adaptive immunity. Recent studies suggest that TLRs activation is likely to contribute to the establishment and maintenance of inflammation in COPD. But most of our knowledge of TLRs has emerged from studies of mice. The contribution of TLRs function to human COPD is less advanced.ObjectiveTo investigate the role of TLRs in the pathogenesis of COPD, we first compared the expression of TLR2 and TLR4 in circulating CD14+ monocytes and neutrophils harvested from clinically stable COPD patients, smokers with normal lung function and never smokers. Then the surface expression of TLR2 and TLR4 was phenotypically characterized on circulating CD14+ monocytes and neutrophils in COPD patients and patients with chronic persistent asthma. In addition, the effects of Th1- type cytokines and corticosteroid on the expression of TLRs in human respiratory epithelial cells were also examined.MethodsThe study population consisted of 30 COPD patients without evidence for acute exacerbation, 21 smokers with normal lung function, 25 healthy non-smokers and 30 patients with persistent asthma. The expression of TLR2, TLR4 surface molecules on human CD14+ monocytes and neutrophils was assessed using FACS analysis by flow cytometry. Spirometries were performed in all participants. Sputum bacterial isolation and serum C-reactive protein (CRP) measurement were performed in COPD patients. Serum tumour necrosis factor-α(TNF-α) and interleukin-8 (IL-8) levels of COPD and asthma patients were measured by means of ELISA. In addition, the human epithelial lung cancer cell line, A549, were stimulated with Interferon-γ(IFN-γ) , TNF-αand dexamethasone singly, or in a combination. Real-time PCR were used to assess TLRs mRNA expression.ResultsClinical study1. CD14+ monocytes expressed significantly higher levels of TLR2 and TLR4 than meutrophils. The expression of TLR2 was positive correlated with the expression of TLR4 on monocytes. The expression of TLR2, and TLR4 as well, in monocytes were positive correlated with that in neutrophils.2. There were 16 clinically stable COPD patients with sputum pathogenic bacterial quantity more than 10~5 CFU/mL among 24 patients whose sputum sample was valid. All the COPD patients had a normal C-reactive protein level.3. Analysis among COPD patients, smokers with normal lung function, and nonsmokers:a. The expression of TLR2 and TLR4 on monocytes in healthy smokers was reduced as compared to nonsmokers ( P<0.05), while in COPD patients the expression was down-regulated more than healthy smokers( P<0.05).b. The expression of TLR2 on neutrophils in healthy smokers was reduced as compared to nonsmokers( P<0.05).The expression of TLR2 on neutrophils showed a tendency to be lower in COPD patients than healthy smokers. There was no significant difference among 3 groups in terms of the expression of TLP4 on neutrophils (P<0.05).c. The expression of TLR2 and TLR4 on monocytes was positively correlated with lung function parameters, including FEV1% predicted and FEV1/FVC( P<0.05). The expression of TLR2 on neutrophils was also positively correlated with the lung function parameters above( P<0.05).4. The TLR2 positive and TLR4 positive cell percent of CD14 monocytes was significantly higher in patients with chronic persistent asthma, than that in healthy controls( P<0.05), while the expression of TLR2 and TLR4 on neutrophils were down regulated in asthma patients. The expression intensities of TLR2 and TLR4 on monocytes showed a tendency to be correlated with serume IgE level in asthma patients.5. Analysis between patients with COPD and patients with asthma:a. Compared with patients with asthma, the expression of TLR2 anf TLR4 on circulating neutrophils was significantly increased in patients with COPD ( P<0.05), while the expression of TLR2 and TLR4 on monocytes was reduced in patients with COPD (P<0.01).b. Compared with patients with asthma, the serum IL-8 level was increased in COPD patients, while the serum TNF-αlevel was reduced in COPD patients.c. The expression of TLR2 and TLR4 on neutrophils were positively associated with serum IL-8 level ( P<0.01). The TLR2 positive and TLR4 positive percent of monocytes were positively associated with serum TNF-αlevel (P<0.01).Laboratory study1. IFN-γsignificantly up regulated the expression of TLR3 on A549 cells (3.78 folds compared with control, P<0.05).2. TNF-αenhanced the expression of TLR2 and TLR4 on A549 cells (1.62 and 1.22 folds compared with control, respectively, P<0.05).3. Dexamethasone significantly up regulated the expression of TLR2 and TLR4 on A549 cells (3.27 and 3.43 folds compared with control, respectively, P<0.05).4. Dexamethasone potentiated TLR2 expression induced by IFN-γor TNF-α(6.25 and 6.09 folds, respectively, P<0.05).5. TLR2 expression was significantly enhanced after stimulation with a combination of TNF-αand IFN-γ(3.66 fold compared with control, P<0.05). Dexamethasone synergistically enhanced TLR2 expression in combination with TNF-αand IFN-γ(29.81 fold compared with control, P<0.05).Conclusion1. CD14+ monocytes expressed significantly higher levels of TLR2 and TLR4 than meutrophils in healthy non-smokers. 2. Smoke depressed the the expression of TLRs on monocytes and neutropils.3. The innate immune response might be depressed in patients with COPD, and the down regulation of TLRs was associated with the disease progression.4. The character of TLRs expression in patients with COPD might play a role in neutrophic inflammation.5. Th-1 type cytokines and corticosteroid regulated the expression of respiratory epithelial cells. Corticosteroid in combination with TNF-αand IFN-γcould synergistically enhance TLR2 expression on respiratory epithelial cells.更多还原