【作者】 沈暘；

【导师】 洪苏玲；

【作者基本信息】 重庆医科大学 ， 耳鼻咽喉科学， 2012， 博士

【摘要】 背景与目的：鼻息肉病(nasal polyposis, NP)是耳鼻咽喉科常见多发疾病。NP的治疗主要包括手术治疗、抗菌素和全身及局部皮质类固醇的应用，但目前NP的疗效并不完全理想，且NP的复发率仍较高。由于NP较高的发病率及其带来的巨大社会负担，NP的防治已逐渐成为一个全球性社会卫生问题。在过去的20年里，各国学者展开了大量关于NP的研究，但其发病机制至今尚未阐明，变应性因素在NP发病机制中的作用尚存争议。目前学术界普遍认为，NP是一个多因素导致的鼻腔鼻窦粘膜慢性炎症性疾病，感染、炎症、变应性因素及解剖畸形等多种因素都可能参与NP的发生发展。Th17细胞是近年来发现的一类不同于Th1、Th2的CD4+T淋巴细胞新亚群，主要通过分泌IL-17A、F等细胞因子发挥促炎作用，在人类RORc为Th17细胞分化生长的关键转录因子，Th17细胞在自身免疫性疾病、慢性炎症性疾病及变应性疾病中均发挥重要作用。调节性T(Treg)细胞是一类在调节机体免疫反应、维持免疫平衡方面具有重要作用的Th细胞亚群，它主要通过细胞接触依赖机制或分泌细胞因子IL-10、TGF-β等发挥作用。Treg细胞功能的异常可导致肿瘤、自身免疫性疾病及变应性疾病的发生，Foxp3为其分化生长的关键转录因子。TH17、Treg细胞均来源于初始T淋巴细胞，其功能和分化过程相互拮抗。正常情况下两者保持相对平衡，Th17/Treg平衡有利于机体免疫稳定状态的维持。既往研究发现，Th17/Treg细胞比率失衡存在于类风湿性关节炎、原发性肾病综合症、炎性肠病等多种慢性炎症性疾病中，提示Th17/Treg失衡可能是慢性炎症性疾病的特征性病理改变，在其发病机制中发挥重要作用。那么，Th17/Treg失衡是否存在于NP中呢？它与变应性因素在NP中的作用又有何联系呢？为探索以上科学问题，我们选择NP患者为研究对象，根据是否伴变应性体质，将其分为atopic NP和non-atopic NP两组，从TH17/Treg平衡入手，研究中国西南地区汉族人群atopic和non-atopic NP的炎症免疫应答特征，以期阐明atopic和non-atopic NP中Th细胞亚型功能失衡的特征，探讨变应性因素在NP中的作用及其可能的机制。本实验将为NP发病机制的研究开辟新的途径，提供新的研究靶点，完善NP发病机制中的Th细胞失衡理论，为其防治策略的建立寻找到新的突破口。第一部分伴和不伴变应性体质鼻息肉患者临床及病理特征对比研究目的：观察伴和不伴变应性体质的鼻息肉患者临床表现和病理特征，分析两者异同及联系，以进一步探讨变应性因素在鼻息肉发病机制中的作用。方法：收集2010年1月～2010年10月在我科住院，根据《欧洲鼻-鼻窦炎、鼻息肉诊疗指南》（EPOS2007）诊断为慢性鼻窦炎伴鼻息肉，行鼻内镜鼻窦手术的成年患者临床资料及术中鼻息肉组织。根据皮肤点刺试验结果，将患者分为伴变应性体质组（atopic NP）和不伴变应性体质组(non-atopic NP)，术前患者主观症状采用视觉类比法(VAS)进行评分；鼻内镜检查结果采用Lanza-Kennedy法评分；鼻窦CT采用Lund-Mackay法评分；鼻息肉组织行HE染色，高倍镜下计数嗜酸性粒细胞数（EOS）并测量基底膜厚度（BM）。结果：共收集鼻息肉成年患者46例（男22例、女24例，年龄23~70岁）；atopic-NP组26例，其中复发鼻息肉6例，伴哮喘3例；non-atopicNP组20例，其中复发鼻息肉3例。与non-atopic NP组相比，atopic-NP组患者的鼻内镜评分、CT评分显著升高；与non-atopic NP组相比，atopic NP组患者的息肉组织中EOS浸润程度及BM增厚程度显著升高；atopic NP组和non-atopic NP组患者年龄、性别、病程、是否复发及症状评分间差异无统计学意义，而伴哮喘的鼻息肉患者仅发现于atopic NP组。结论：伴变应性体质鼻息肉病，累及范围更广，病变程度更重；变应性因素可能通过促进EOS浸润及粘膜重建，促进鼻息肉发生发展；在鼻息肉病围手术期综合治疗中，有必要区分患者是否伴变应性体质，对变应体质者除常规手术药物治疗外，还应积极抗变态反应治疗。是否伴有变应性体质可作为鼻息肉分型分类的指标之一，指导临床治疗。第二部分伴和不伴变应性体质鼻息肉患者Th17/Treg表达失衡的实验研究目的：通对检测NP患者外周血中Th17和Treg细胞比率及息肉组织中Th17和Treg细胞在RNA、蛋白水平的相关因子，观察atopic和non-atopic NP两组患者外周和鼻腔鼻窦病变局部Th17/Treg平衡状态及两组间异同，旨在探讨Th17/Treg细胞失衡在NP发病机制中的意义，及NP中变应性因素与Th17/Treg平衡间的相互关系。方法：采用流式细胞术检测两组NP患者外周血Th17、Treg细胞比率，采用real-time PCR检测息肉组织中RORc、Foxp3表达，采用ELISA检测息肉组织中IL-17、 TGF-β、IL-10及INF-γ、 IL-4、 IL-5水平，并对外周血Th17、Treg细胞比率与息肉组织中相关因子分析相关性分析，对Th17、Treg及其相关因子与Th1、Th2型细胞因子进行相关性分析。结果：NP患者外周血中Th17细胞比率及息肉组织中RORc、IL-17水平较对照组显著升高，且atopic NP组与non-atopic NP组间差异显著，具有统计学意义； NP患者外周血中Treg细胞比率及息肉组织中Foxp3、TGF-β水平较对照组显著降低，且atopic NP组与non-atopic NP组间差异显著，具有统计学意义，而IL-10水平显著升高。NP患者外周血Th17细胞比率与息肉组织中RORc、IL-17水平正相关，外周血Treg细胞比率与息肉组织中Foxp3、TGF-β水平正相关；Treg及其相关因子与息肉组织中Th1、Th2型细胞因子呈负相关，而Th17细胞及其相关因子与息肉组织中Th1、Th2型细胞因子相关性无统计学意义。结论：Th17细胞数量和功能的过度亢进及Treg细胞数量和功能的过度抑制存在于NP中，Th17/Treg失衡在NP发病机制中扮演重要角色；NP患者外周血及息肉组织中Th17/Treg消长一直， Th17/Treg平衡在机体外周和局部的免疫应答中均发挥作用，鼻腔鼻窦局部的Th17、Treg细胞大部分来源于外周血。Atopic NP存在更为严重的Th17/Treg失衡和更为严重的临床病理学特征，变应性因素可能通过促进Th17/Treg失衡推动NP的发生发展。NP患者外周血中Treg细胞数量与息肉组织中Th1、Th2细胞型细胞因子成负相关，NP中Th1/Th2失衡可能由于Treg对其免疫抑制功能受损所致。第三部分尘螨变应原体外刺激对鼻息肉患者外周血Th17、Treg细胞的影响目的：通过大剂量尘螨特异性变应原(HDM)短期体外刺激培养atopic和non-atopic NP患者外周血单个核细胞(PBMC)，观察变应原体外刺激对两组NP患者PBMC中TH17/Treg的不同影响，分析两组间变化差异及其与Th1、Th2型细胞因子的相关性，以期在前部分实验基础之上进一步阐述两组患者TH17/Treg功能的异同，体外证实TH17/Treg失衡在NP发病机制中的作用及其与变应性因素的相互关系。方法：选用大剂量HDM体外刺激培养atopic和non-atopic NP患者PBMC48h，采用流式细胞术检测两组NP患者PBMC中Th17、Treg细胞比率，采用real-time PCR检测外周血T细胞中RORc、Foxp3表达，采用ELISA检测外周血T细胞中IL-17、TGF-β、IL-10及INF-γ、IL-4、 IL-5水平，并分析Th17、Treg与Th1、Th2型细胞因子的相关性。结果：HDM+PHA刺激使atopic NP组患者外周血中Th17细胞比率及其在RNA、蛋白水平的相关因子RORc、IL-17较non-atopic NP组及对照组显著升高，而non-atopic NP组与对照组间差异无统计学意义。此外，在atopic NP组，HDM+PHA刺激使Th17细胞比率及其相关因子较PHA刺激显著升高。HDM+PHA刺激使atopic NP组和non-atopic NP组患者外周血中的Treg细胞比率及其在RNA、蛋白水平的相关因子Foxp3、TGF-β均较对照组显著降低，且两组间差异显著，而IL-10水平显著升高。此外，在atopic组，HDM+PHA刺激使Treg细胞比率及其相关因子较PHA刺激显著降低，而在对照组，HDM+PHA刺激使Treg细胞比率及其相关因子较PHA刺激显著升高。NP患者PBMC中Treg细胞比率在PHA和HDM+PHA刺激后均与上清液中Th1、Th2型细胞因子(INF-γ、 IL-4、 IL-5)水平呈负相关，而Th17细胞比率与Th1、Th2型细胞因子水平相关性无统计学意义。结论：在致敏NP患者体内HDM可诱导变应原特异性的Th17免疫应答上调，揭示了Th17细胞在气道变应性炎症中的重要作用，证实了变应性因素可通过Th17细胞在NP中发挥作用。诱导Treg细胞的增殖分化可能在正常机体针对变应原的免疫应答中具有重要意义，这一机制在NP患者特别是atopic NP患者体内受损，导致Treg细胞免疫调节功能下调，对Th1/Th2的免疫抑制下调，机体免疫失衡，推动疾病发展。更多还原

【Abstract】 Background and Objectives Nasal polyposis (NP), commonlyencountered in clinical otorhinolaryngology, is a chronic inflammatorydisease of the nasal cavity and sinus. Nowadays the treatment outcomes ofantibiotics, steroids, and surgery for NP are unsatisfactory, and therecurrence rate remains high. NP has become a more and more importantsocial issue because of its high incidence and considerable economic burden.Over the last2decades, the pathogenesis of NP has been studied widely, butit is not clearly understood yet, and the role of atopy in the etiology andpathogenesis of NP is still a controversial issue. Nowadays NP is consideredto be a multifactorial disease and generally represent a subset of chronicinflammation of the mucous membrane in the paranasal sinus, which isdistinct from chronic rhinosinusitis without NP. A variety of allergic,infectious, inflammatory and anatomical factors are known to be involved inthe origin of NP.Recently, Th17cells and regulatory T (Treg) cells have been describedas two subsets distinct from Th1and Th2cells. Th17cells are characterizedby their preferential production of interleukins (IL)-17A and F and requireretinoid orphan nuclear receptor (RORc) as a key transcription factor for their differentiation in humans; Th17cells play critical roles in thedevelopment of autoimmunity, inflammation and allergic reaction. Treg cellsexpressing the forkhead/winged helix transcription factor (Foxp3) arethought to maintain immunological self-tolerance and have ananti-inflammatory role that functions by contact-dependent suppression orby releasing anti-inflammatory cytokine IL-10and transforming growthfactor (TGF-β). Th17and Treg not only exhibit opposite functions in theimmune response but also share reciprocal development pathways. Theimbalance of Th17/Treg still exists in patients with juvenile arthritis,primary nephritic syndrome and inflammatory bowel disease, suggestingthat the imbalance of Th17/Treg may be a feature of pathologicinflammatory disease and play an important role in the pathogenesis ofchronic inflammatory disease.In this study, we divided NP patients according atopy or non-atopy, andthe TH17/Treg balance was designed as the entry point. We evaluated theimmune characteristics of NP patients with atopy or not, in order todetermine the different features of Th cells dysfunction in NP patients withatopy or not, and discuss the possible correlation between atopy and NP. Thisresearch will open a new avenue, provide new targets for the research of NP,and improve the Th cell imbalance theory in the pathogenesis of NP. Thisresearch can provide a new breakthrough for the establishment of controlstrategies for NP. Part one The difference in some clinical and pathologicalcharacteristics between nasal polyposis patients with and without atopyObject: we aimed to observe the clinical characteristics andhistological features of atopic and non-atopic NP patients, and then analyzethe differences and correlations between two groups, finally investigate therole of atopy in the pathogenesis of nasal polyposis.Method: NP patients in our department from2010.1-2010.10werecollected. The diagnosis of NP was made according to the current EuropeanEAACI Position Paper on Rhinosinusitis and Nasal Polyps and Americanguidelines. NP patients were divided into atopic or non-atopic NP groupbased on skin prick test (SPT). Clinical data and polyp tissue were collected.Symptom scores were assessed according to a visual analog scale (VAS).The preoperative computed tomography (CT) scans were graded accordingto the classification by Lund and Mackay. The preoperative nasal endoscopyscores were graded according to the classification by Lanza and Kennedy.The polyp tissue sections were stained with hematoxylin and eosin (H-E).Maximal basement membrane (BM) thickness and the number ofeosinophils were detected at a magnification of×400.Results: Forty-six patients (24males,22females;26atopic,20non-atopic) between23and70years of age were included. Endoscopy score,and CT score were significantly higher in the atopic group than in the non-atopic group. However, no statistically significant differences werefound between two groups from the standpoint of symptom score, age, sex,duration of disease or recurrence, and asthma was only noted in the atopicgroup. Histologically, the mean number of eosinophils in the atopic groupwas significantly increased compared to that of the nonatopic group. TheBM was statistically thicker in atopic compared to nonatopic patients.Conclusion: In atopic NP patients, there were more severe clinical andhistological features. Atopy may aggravate NP by promoting the infiltrationof EOS in polyp tissue. In the treatment of NP, it is necessary to distinguishthe atopic NP patients from non-atopic ones, and provide them activelyanti-allergy treatment. Atopic constitution can be used as a sub-classificationfor NP to guide clinical treatment.Part two Impaired balance of Th17/Treg in patients with nasalpolyposisObject: To assess whether the balance of Th17/Treg is disrupted inpatients with NP, we evaluated the distribution of Th17and Treg cells amongperipheral blood mononuclear cells (PBMCs) in atopic NP patients,non-atopic NP patients and controls.Method: We evaluated the distribution of Th17and Treg cells amongperipheral blood mononuclear cells (PBMCs) in atopic NP patients,nonatopic NP patients and controls. Then we determined mRNA levels of RORc, Foxp3and protein levels of IL-17, TGF-β and IL-10in polyp tissueamong the three groups. Finally, we investigated the correlation betweenTh17, Treg and Th1, Th2related cytokines (INF-γ, IL-4, IL-5).Results: The results demonstrated that both atopic and non-atopicpatients with NP revealed significantly increased Th17proportion anddecreased Treg proportion in PBMCs, as well as significantly increasedRORc and IL-17levels and decreased Foxp3and TGF-β levels in polyptissue. Furthermore, these differences were significant between atopic andnon-atopic groups. The frequency of Treg in PBMCs was found to benegatively correlated with Th1and Th2cytokines in polyps.Conclusion: These results indicated that an impaired balance ofTh17/Treg existed in patients with NP and was more severe in atopic patients,suggesting that the imbalance of Treg Th17may play an important role inthe development of NP and that atopy may aggravate NP by promoting theimbalance of Th17/Treg. The imbalance of Th1/Th2in NP may be ascribedto decreased regulation of them by Treg.Part three Allergen induced Th17and Treg cells response in theperipheral blood mononuclear cells (PBMCs) of patients with nasalpolyposisObject: This study aimed to investigate the population and function ofperipheral Th17and Treg cells in response to HDM allergen in NP patients, and evaluate the possible correlation between Th17/Treg cells and atopy, toexplore the role of atopy in the pathogenesis of NP.Methods: Peripheral blood mononuclear cells (PBMC) obtained fromatopic NP patients, non-atopic NP patients, and controls were stimulated byphytohemagglutinin (PHA) or house dust mite extracts (HDM) plus PHA.The resulting frequency of Th17and Treg cells was detected by flowcytometry and the expression of RORc and Foxp3was measured byreal-time PCR. Then the supernatants were assayed for IL-17, TGF-β、IL-10，INF-γ, IL-4and IL-5by specific ELISAs.Results: The population and function of Th17cells in allergenstimulated PBMCs were significantly higher in atopic NP patients. Inaddition, in atopic group, HDM+PHA stimulation induced significantincrease of Th17population and IL-17production versus those in PHAstimulated ones. However, the frequency of Th17cells was not correlatedwith Th1, Th2cytokine productions. Both atopic and non-atopic NP patientsrevealed significantly decreased frequency of Treg cells and Foxp3level inallergen stimulated PBMCs, as well as significantly decreased TGF-β levelin culture supernatants. The decrease was even more striking in atopic group.Also, there were significant negative correlations between Treg cells andINF-γ, IL-4, IL-5.Conclusion: Th17and Treg immunity is involved in the systemicimmune responses to allergen in atopic NP and atopy may aggravate NP by stimulating the increase of Th17population and decrease of Treg production.Patients with NP had a defective regulatory T cell response after allergenstimulation which was related to the excessive Th1, Th2responses tospecific allergen.更多还原