【作者】 李海军；

【导师】 肖伟；

【作者基本信息】 山东大学 ， 内科学， 2011， 硕士

【摘要】 研究背景T辅助细胞(Th细胞)是天然免疫和获得性免疫连接的纽带,幼稚CD4+T淋巴细胞接受固有免疫信号后向不同方向分化进而调节细胞免疫和体液免疫。Th17细胞是新发现的T辅助细胞的成员,因其特异性的分泌IL-17而得名,类似于Th1与Th2之间的平衡,Th17细胞与Treg之间也存在重要平衡。Th17可以通过分泌IL17、IL-21、IL-22、IL-6等募集中性粒细胞和巨噬细胞,进一步放大炎症反应,而在炎症性疾病中发挥致病作用。而Treg通过直接接触或分泌细胞因子(如IL-10, TGF-β)等抑制炎症反应,维持免疫自稳。两者功能相互拮抗,两者之间平衡维持或破坏对于免疫稳定或炎症性疾病的发病具有重要意义。大量研究已证实,Th17/Treg失衡在许多炎症性疾病如鼻息肉、狼疮性肾炎、扩张性心肌病、类风湿性关节炎、炎症性肠病中发挥致病作用。而COPD作为一种持续的慢性炎症性疾病,Th17/Treg失衡可能在其发病研究中具有重要意义。目的本研究旨在探讨COPD患者外周血和诱导痰中Th17细胞和Treg细胞平衡关系,分析其在COPD发病中的作用。方法选取稳定期COPD患者21例,健康吸烟者(healthy smokers, HS)21例,健康非吸烟者(healthy non-smokers, HNS)21例,所有受试者均进行肺通气功能检查,然后采集空腹静脉血及诱导痰。实时定量PCR技术检测诱导痰细胞及外周血单个核细胞(peripheral blood mononuclear cell, PBMC)中Th17及Treg特征性转录调控因子RORC2及Foxp3 mRNA表达水平；流式细胞分析技术检测受试者外周血Th17细胞及Treg细胞比例；酶联免疫吸附试验(enzyme-linked immunoadsordent assay, ELISA)检测诱导痰及外周血Th17及Treg相关细胞因子(包括IL-17、IL-10、TGF-β、IL-6)。结果1. COPD患者诱导痰中存在Th17及Treg转录调控因子及相关细胞因子表达变化在转录调控因子水平,COPD患者RORC2 mRNA较健康吸烟组与健康非吸烟组显著升高；Foxp3 mRNA水平较健康非吸烟对照下降,较健康非吸烟组无显著统计学差异; RORC2 mRNA水平与Foxp3mRNA水平的比值较另外两组显著升高。在细胞因子水平,COPD患者诱导痰中IL-17水平较另外两组升高,IL-10水平无显著变化。TGF-β及IL-6水平均升高。2. COPD患者外周血Th17及Treg细胞比例、转录调控因子及细胞因子水平变化在细胞水平,Th17细胞比例与Treg细胞比例在COPD与健康吸烟对照外周血中均升高,Thl7细胞比例与Treg比例之间的比值在三组之间无明显差异。在转录调控因子水平,COPD患者PBMC RORC2 mRNA与Foxp3 mRNA表达均升高,RORC2 mRNA水平与Foxp3mRNA水平的比值较另外两组显著升高。在细胞因子水平,COPD患者外周血IL-10水平较另外两组均升高,IL-17水平较两外两组有升高趋势,但未达到显著统计学差异标准(P=0125)。在COPD患者外周血,TGF-β及IL-6水平升高。3.重要相关性分析(1)吸烟者诱导痰细胞RORC2表达水平与呈吸烟量正相关；(2)诱导痰细胞RORC2表达水平与痰TGF-p呈正相关；(3)痰IL-17水平与痰IL-6水平呈正相关；(4)诱导痰细胞RORC2、IL-17及TGF-β与FEV1 (pred%)及FEV1/FVC (%)分别呈显著负相关。结论本研究分别探讨了Th17与Treg在COPD局部气道和外周血的平衡关系及其意义。在COPD患者局部气道,Th17/Treg平衡向Th17方向偏移,抑制炎症作用不足,RORC2表达及活性上调,IL-17分泌增多,IL-17与FEVlpred%和FEV1/ FVC分别呈负相关,其可以通过促进气道炎症、黏液高分泌及气道重塑参与COPD发病。在外周血,虽然在细胞水平Th17与Treg等比例上调,但在转录调控因子水平仍存在Th17/Treg失衡,系统性炎症仍低水平持续。TGF-β与IL-6是Th17与Treg平衡的重要调定点,其在COPD复杂内环境下的精确作用有待进一步研究。更多还原

【Abstract】 BackgroundRecently, regulatory T cells (Tregs) and Th17 cells have been identified as two distinct CD4+T cell subsets from Thl and Th2 cells. Tregs express CD4 and CD25 on the cell surfaces, and they are identified by the nuclear expression of the characteristic transcription factor forkhead box P3 (FoxP3), while Th17 cells express retinoic acid-related orphan receptor C2 (RORC2). Tregs can limit the strength of the immune response of inflammatory cells by contact-dependent suppression or releasing anti-inflammatory cytokine interleukin-10(IL-10) and transforming growth factorβ(TGF-β), and have been shown to play an essential role in the induction and maintenance of peripheral tolerance, while Th17 cells possess very strong inflammatory properties by secretion of proinfammatory cytokines IL-17, IL-22, IL-23 and et al, and have been shown to play an critical role in a number of inflammatory diseases. So the imbalance of Th17 cells and Tregs(Th17/Treg imbalance) is supposed to play a pathogenic role in many inflammatory and autoimmune diseases, and this has been confirmed in lupus nephritis psoriasis, rheumatoid arthritis, idiopathic dilated cardiomyopathy, nasal polyposis, and et al. As a definite inflammatory disease, COPD may also be involved in the pathogenesis of this imbalance.ObjectiveThe objective of this study was to evaluate whether the Th17/Treg balance was broken in patients with COPD in both peripheral blood and local airways as well as to explore its significance in the pathogenesis of COPD.MethodsThe present study involved 21 patients with COPD,21 healthy smokers with normal lung function(HS)and 21 healthy non-smokers(HNS). In the peripheral blood, flow cytometry analysis was used to detect Th17 and Treg frequencies, however, in the sputum, there were not enough cells for flow cytometry analysis, and instead, we used real-time PCR to detect their characteristic transcription regulators, RORC2 and Foxp3 to analyze Th17 and Treg expression level. The concentrations of Th17 and Treg associated cytokines(IL-17, IL-10, TGF-β, IL-6) were detected in both serum and sputum with enzyme-linked immunoadsordent assay(ELISA).Results1. In the sputum from COPD patients, Th17/Treg was imbalanced at the characteristic transcription regulator level as well as cytokine level The level of RORC2 in sputum cells from COPD patients was higher than those from both HS group and HNS group, and the RORC2 mRNA was also higher in HS group than that of HNS group. However, up-regulation of Foxp3 mRNA was seen only in HS group. Sputum IL-17 was also upregulated while IL-10 failed.2. In the peripheral blood from COPD patients, Th17/Treg was also imbalanced at the characteristic transcription regulator levelIn the peripheral blood, Th17 cell frequencies and Treg cell frequencies were both up-regulated COPD patients. The level of RORC2 in PBMC from COPD patients, RORC2 mRNA and Foxp3 mRNA were unregulated in COPD group, and the ratio of RORC2 to Foxp3 was also higher than those in HS group and HNS group. Plasma IL-17 showed an increasing tendency (P=0.125) when compared with healthy non-smokers group, while a higher IL-10 concentration was seen.3. In COPD patients, higher levels of TGF-βand IL-6 were seen in both sputum and plasma when compared with those from HNS group4. Significant correlations(1) In smokers, the level of RORC2 insputum cells was positively correlated with smoking frequency.(2) The level of RORC2 in sputum cells was positively correlated with sputum TGF-β.(3) The level of sputum IL-17 positively correlated with that of sputum IL-6. (4) Both the level of RORC2 in sputum cells and sputum IL-17 were negatively correlated with FEV1 (pred%) and FEV1/FVC (%) separately.ConclusionOur study explored Thl7/Treg balance status in both airways and peripheral blood in COPD patients. In the local airways, Thl7/Treg was imbalanced, and inhibition by Treg was not sufficient, thus RORC2 activities and IL-17 was upregulated, which might play pathogenic roles in promoting airway inflammation, mucus hypersecretion as well as airway remodeling, and we found a negative correlation between IL-17and FEV1pred% or FEV1/FVC. in the peripheral blood, there was a relatively balanced status in Thl7/Treg cell frequencies, however, Thl7/Treg was imbalanced at the characteristic transcription regulator level, and this imblance might play a role in systemic damage. TGF-βand IL-6 were important points in regulating Th17/Treg balance status, however, the exact mechanism needed to be explored furtherly.更多还原