【作者】 廖竞；

【导师】 农光民；

【作者基本信息】 广西医科大学 ， 儿科学， 2014， 博士

【摘要】 支气管哮喘（哮喘）是由多种炎症细胞（嗜酸性粒细胞、肥大细胞、T淋巴细胞、中性粒细胞等）、气道结构细胞（气道上皮细胞、平滑肌细胞、成纤维细胞）以及多种细胞因子共同参与的气道慢性炎症性疾病。哮喘发病机制复杂，至今尚未完全阐明。气道慢性炎症是哮喘的本质，研究哮喘气道炎症发生、发展机制对于哮喘防治具有重要临床指导意义。随着对哮喘气道炎症的深入研究，人们对哮喘的认识已不再局限于嗜酸性粒细胞气道炎症，而更强调哮喘是多种细胞共同参与的气道慢性炎症。目前研究显示哮喘的气道炎症存在不同的亚型，可分为嗜酸细胞性哮喘（eosinophilicasthma，EA）和非嗜酸细胞性哮喘（non-eosinophilic asthma，NEA），其中半数以上的非嗜酸细胞性哮喘为中性粒细胞性哮喘（neutrophilic asthma，NA）。但哮喘气道中性粒细胞增多的机制目前尚未完全阐明。辅助性T淋巴细胞(T helper cell，Th细胞)在哮喘气道慢性炎症中发挥着重要的免疫调节作用。Th2细胞占优势的Th1/Th2细胞失衡被认为是变应性哮喘气道嗜酸细胞性炎症形成及发展的免疫学基础。但有研究发现非变应性哮喘患者存在不依赖于Th2细胞免疫学机制介导的气道中性粒细胞炎症。近年来发现Th17细胞及其效应因子IL-17在支气管哮喘等变态反应性疾病中发挥重要作用， IL-17具有强大的募集、活化中性粒细胞的作用，可能是导致气道中性粒细胞增多的机制之一。但Th17细胞在不同气道炎症类型哮喘发病中的作用是否相同，在中性粒细胞性哮喘发病机制中是否存在Th17细胞分化优势，鲜有报道。我们前期动物实验通过建立中性粒细胞哮喘小鼠模型进行研究，结果显示中性粒细胞哮喘小鼠存在强烈的Th17细胞免疫、中度Th2细胞免疫反应，Th17细胞通过IL-17介导哮喘小鼠中性粒细胞性气道炎症的发生，并通过上调转录因子RORγt促进Th17细胞分化，中性粒细胞哮喘小鼠体内已分化的Th17细胞在机体高IL-7环境下，依赖JAK/STAT5信号途径激活维持其存活状态。但在人类中性粒细胞性哮喘发病机制中是否存在同样的Th17细胞分化优势是本课题所要研究的第一个问题。糖皮质激素作为治疗哮喘最有效的抗炎药物主要通过减少T淋巴细胞活化及抑制细胞因子的产生减轻气道炎症反应。对于多数哮喘患者以吸入糖皮质激素（ICS）为基础的规范化哮喘治疗可达到哮喘控制，但仍有部分患者并没有取得预期的效果，少数患者症状持续存在、甚至恶化，随着对哮喘气道炎症的深入研究，发现激素治疗效果欠佳的哮喘患者多为中性粒细胞哮喘。近年有研究发现，糖皮质激素不能抑制Th17细胞及其效应因子IL-17介导的气道中性粒细胞炎症，但亦有研究显示糖皮质激素治疗可下调IL-17表达，我们前期动物实验研究亦发现地塞米松可通过下调中性粒细胞哮喘小鼠Th17细胞特异性转录因子RORγt表达水平，降低Th17细胞数量及其相关细胞因子IL-17水平，部分减轻哮喘小鼠气道中性粒细胞炎症。但地塞米松对Th17细胞存活延长的状态及气道中性粒细胞凋亡率无明显影响。目前，对于糖皮质激素在Th17细胞介导的哮喘气道炎症中的作用存在不同的研究结果。我们将在探讨Th17细胞与儿童中性粒细胞性哮喘气道炎症相关性的基础之上，进一步探讨糖皮质激素对Th17细胞介导的儿童中性粒细胞性哮喘气道炎症的作用。本研究将初步阐明Th17细胞在儿童中性粒细胞性哮喘发病中的作用，为儿童中性粒细胞性哮喘的治疗寻求新线索及提供理论依据，有助于哮喘的个体化治疗。研究分为两部分进行：第一部分Th17细胞及相关细胞因子与儿童中性粒细胞性哮喘气道炎症相关性分析目的探讨Th17细胞及相关细胞因子与儿童中性粒细胞性哮喘气道炎症的相关性。方法随机选取28例未应用糖皮质激素治疗急性发作期哮喘患儿，进行肺功能测定、诱导痰细胞分类计数，并根据诱导痰液细胞学分类分成三组：嗜酸细胞性哮喘（EA组）12例；中性粒细胞哮喘（NA组）10例；非嗜酸细胞非中性粒细胞性哮喘（NEA+NNA组）共6例，并选取健康对照组(HC组)10例，采用流式细胞术检测外周血Th17、Th2细胞及Th17细胞上Ki-67、STAT5、BCL-2的表达；实时荧光定量聚合酶链反应（Real-time PCR）检测外周血单个核细胞RORγt-mRNA的表达；ELISA方法检测痰上清液、血浆、经PMA刺激的PBMC培养上清液IL-17浓度、痰上清液IL-8、IL-5浓度，并进行相关性分析。结果1、NA组外周血Th细胞中Th17细胞百分比较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组Th17细胞百分比较HC组增高(p<0.01)，EA组与NEA+NNA组间Th17细胞百分比比较差异无统计学意义(p>0.05)；EA组外周血Th细胞中Th2细胞百分比较NA组、NEA+NNA组、HC组均增高(p<0.01)，NA组、NEA+NNA组Th2细胞百分比较HC组增高(p <0.01)，NA组与NEA+NNA组间Th2细胞百分比比较差异无统计学意义(p>0.05)；2、NA组外周血Th17细胞Ki-67表达阳性率较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组Th17细胞Ki-67表达阳性率较HC组增高(p<0.01)，EA组与NEA+NNA组间Th17细胞Ki-67表达阳性率比较差异无统计学意义(p>0.05)；NA组外周血Th17细胞STAT5表达阳性率较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组Th17细胞STAT5表达阳性率较HC组增高(p<0.01)，EA组与NEA+NNA组间Th17细胞STAT5表达阳性率比较差异无统计学意义(p>0.05)；NA组外周血Th17细胞BCL-2表达阳性率较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组与HC组间Th17细胞BCL-2表达阳性率比较差异无统计学意义(p>0.05)；3、NA组外周血单个核细胞RORγt-mRNA表达较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组RORγt-mRNA表达较HC组增高(p<0.01)，EA组与NEA+NNA组间RORγt-mRNA表达比较差异无统计学意义(p>0.05)；4、NA组痰上清液IL-17浓度较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组痰上清液IL-17浓度较HC组增高(p <0.01)，EA组与NEA+NNA组间痰上清液IL-17浓度比较差异无统计学意义(p>0.05)；EA组痰上清液IL-5浓度较NA组、NEA+NNA组、HC组均增高(p <0.01)，NA组、NEA+NNA组痰上清液IL-5浓度较HC组增高(p <0.01)，NA组与NEA+NNA组间痰上清液IL-5浓度比较差异无统计学意义(p>0.05)；NA组痰上清液IL-8浓度较EA组、NEA+NNA组、HC组均明显增高(p <0.01)，EA组、NEA+NNA组痰上清液IL-8浓度较HC组增高(p <0.01)，EA组与NEA+NNA组间痰上清液IL-8浓度比较差异无统计学意义(p>0.05)；5、NA组、EA组、NEA+NNA组与HC组间血浆IL-17浓度比较差异无统计学意义(p>0.05)，但经PMA刺激培养后，NA组PBMC培养上清液IL-17浓度较EA组、NEA+NNA组、HC组均明显增高(p<0.01)，EA组、NEA+NNA组PBMC培养上清液IL-17浓度较HC组增高(p<0.01)，EA组与NEA+NNA组间PBMC培养上清液IL-17浓度比较差异无统计学意义(p>0.05)；同时，经PMA刺激培养后，NA组、EA组、NEA+NNA组PBMC培养上清液IL-17浓度较血浆IL-17浓度均明显增高(p均<0.01)，HC组PBMC培养上清液IL-17浓度与血浆IL-17浓度比较差异无统计学意义(p>0.05)。6、哮喘患儿诱导痰NEU百分比与外周血Th细胞中Th17细胞比例及痰上清IL-17、IL-8、PBMC培养上清液IL-17浓度呈明显正相关；哮喘患儿诱导痰NEU百分比与FEV1%pred、PEF%pred呈明显负相关；哮喘患儿诱导痰EOS百分比与外周血Th细胞中Th2细胞比例及痰上清IL-5浓度呈明显正相关。结论1、Th17细胞和Th2细胞均不同程度共同参与儿童哮喘的发病。儿童中性粒细胞性哮喘外周血Th17细胞、特异性转录因子RORγt表达及痰液IL-17水平均明显高于嗜酸细胞性哮喘及非嗜酸细胞非中性粒细胞性哮喘，并且与诱导痰中性粒细胞比例呈正相关。提示Th17细胞可能通过IL-17介导中性粒细胞性哮喘气道炎症的发生。2、儿童中性粒细胞性哮喘体内已分化的Th17细胞存活延长，可能与JAK/STAT5信号途径激活有关。第二部分吸入糖皮质激素对Th17细胞介导的儿童中性粒细胞性哮喘气道炎症的影响目的探讨吸入糖皮质激素对Th17细胞介导的儿童中性粒细胞性哮喘气道炎症的影响方法第一部分研究对象中的中性粒细胞性哮喘患儿(NA组)10例，在医师指导下规则吸入糖皮质激素治疗3个月后，进行肺功能测定、诱导痰细胞分类计数；采用流式细胞术检测外周血Th17细胞及Th17细胞上Ki-67、的表达；实时荧光定量聚合酶链反应（Real-time PCR）检测外周血单个核细胞RORγt-mRNA的表达；ELISA方法检测痰上清液、PBMC培养上清液IL-17浓度。结果1、NA组规则吸入糖皮质激素治疗后FEV1%pred、PEF%pred均较治疗前明显增高(均p <0.01)，但仍低于健康对照组（P均<0.05）；2、NA组规则吸入糖皮质激素治疗后诱导痰NEU百分比较治疗前明显降低(p<0.01)，但仍高于健康对照组（P<0.05）；3、NA组规则吸入糖皮质激素治疗后外周血Th17细胞及Th17细胞Ki-67表达均较治疗前明显降低(均p<0.01)，但仍高于健康对照组（均P<0.01）；4、NA组规则吸入糖皮质激素治疗后外周血单个核细胞RORγt-mRNA表达较治疗前明显降低(p<0.01)，与健康对照组比较差异无统计学意义(p>0.05)；5、NA组规则吸入糖皮质激素治疗后痰上清液及PBMC培养上清液IL-17浓度均较治疗前降低(分别为：p<0.05，p <0.01)，但仍高于健康对照组（均P <0.01）。结论吸入糖皮质激素可通过下调特异性转录因子RORγt的表达水平，在一定程度上降低外周血Th17细胞及痰液IL-17水平，减轻Th17细胞介导的儿童中性粒细胞性哮喘气道炎症。更多还原

【Abstract】 Bronchial asthma (Asthma) is one of the most common chronic airwaydiseases worldwide. Airway inflammation plays a key role in the pathogenesis ofasthma. Many cells (such as eosinophils, mast cells, T lymphocytes, neutrophils,and airway epithelial cells, etc.) and cellular elements are involved in thepathogenesis of airway inflammation in asthma.Airway inflammation witheosinophils is accepted as a fundamental characteristic of allergic asthma.However, current studies show there are different subtypes in the airwayinflammation of asthma. According to phenotype of airway inflammation,asthma can be classified into different subtypes: eosinophilic asthma (EA) andnon-eosinophilic asthma（NEA）, and more than half of non-eosinophilic asthmaare characterized by neutrophilic asthma. But the pathogenetic mechanisms ofneutrophilic asthma are not well understood now.T cells are critical to thepathogenesis of asthma. Typically, it is known that Th2cells can promoteeosinophilic airway inflammation in atopic asthma,but recent studies suggestthere are differences in the immunopathology between atopic asthma andnonatopic asthma.Recently, a new T helper cell subset is identified, namely, Th17cells, which characterized by the secretion of IL-17and the expression ofretinoic acid-related orphan receptor γt (RORγt), can promote neutrophilicinflammation.Some studies show that Th17cells and IL-17also seem to beinvolved in asthma pathogenesis.What is the role of Th17cells in neutrophilicasthma? Our previous animal studies have shown that both Th17cells and Th2cells are involved in the pathogenesis of neutrophilic asthma and eosinophilicasthma in mice. Th17cells and IL-17may mediate neutrophilic airwayinflammation in the mouse model of neutrophilic asthma by up-regulatingRORγt expression. And Th17cells may be maintain their survival throughactivation of the JAK/STAT5signaling pathway. However, whether Th17-drivenimmunity contributes to the pathogenesis of neutrophilic asthma in human isunclear,and remains to be investigated.The efficacy of inhaled glucocorticosteroids in reducing airwayinflammation and the symptoms of asthma has led to their widespread use asinitial therapy in the treatment of asthma. Although inhaled glucocorticosteroidsare effective for most of asthmatic patients, it is uncertain whether their use canreduce neutrophilic airway inflammation in some patients with neutrophilicasthma.Recent studies suggest that Th17cell-mediated airway inflammation issteroid resistant. On the contrary,some researches show that glucocorticosteroidscan decrease the level of IL-17in asthmatic patients.What is the effect ofglucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation inneutrophilic asthma? Our previous animal studies have shown thatDexamethasone can reduce Th17cell-mediated neutrophilic airwayinflammation in the mouse model of neutrophilic asthma by decreasingpercentage of Th17cells, level of IL-17and down-regulating RORγt expression.And Dexamethasone has no significant effect on apoptosis of airway neutrophils and survival of Th17cells.But the effect of glucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation in patients with neutrophilic asthmaremains to be investigated. To better understand the role of Th17cells inasthma may improve the understanding of the pathogenesis of asthma.In thisstudy,we will explore the relationship between Th17cells and neutrophilicairway inflammation in childhood neutrophilic asthma, and identify the possiblerole of Th17cells in pathogenesis of neutrophilic asthma in children. Our studywas divided into two parts.Part oneRelationship between Th17cells and neutrophilic airwayinflammation in childhood neutrophilic asthmaObjective To explore relationship between Th17cells and neutrophilicairway inflammation in childhood neutrophilic asthma.Methods Twenty-eight children with exacerbated asthmatics withoutusing any corticosteroids were divided into three groups: eosinophilic asthmagroup (EA group)(n=12), neutrophilic asthma group (NA group)(n=10) andnon-eosinophilic asthma and non-neutrophilic asthma (NEA+NNA group)(n=6) according to induced sputum cytology.Ten healthy children were recruited ashealthy control group (HC group)(n=10). Th17cells,Th2cells in peripheralblood and Ki-67,STAT5, BCL-2expressed in Th17cells were detected by flowcytometry. Expression of RORγt-mRNA in PBMC was detected by Real-timePCR.Concentrations of IL-17, IL-5and IL-8in induced sputum supernatant, aswell as concentrations of IL-17in plasma and in the culture supernatant fromPMA-stimulated PBMC were measured by ELISA Results1.The percentage of Th17cells in peripheral blood wassignificantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01), and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05).The percentage of Th2cellsin peripheral blood was significantly higher in EA group than in NA group,NEA+NNA group and HC group (both P<0.01), and was significantly higher inNA group and NEA+NNA group than in HC group (both P<0.01), but there wasno significant difference between NA group and NEA+NNA group (P>0.05).2.The expression level of Ki-67in Th17cells was significantly higher in NAgroup than in EA group, NEA+NNA group and HC group (both P<0.01). andwas significantly higher in EA group and NEA+NNA group than in HC group(both P<0.01), but there was no significant difference between EA group andNEA+NNA group (P>0.05). The expression level of STAT5in Th17cells wassignificantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01). and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05). The expression level ofBCL-2in Th17cells was significantly higher in NA group than in EA group,NEA+NNA group and HC group (both P<0.01),and there was no significantdifference among EA group,NEA+NNA group and HC group (P>0.05).3. Theexpression level of RORγt-mRNA in PBMC was significantly higher in NAgroup than in EA group, NEA+NNA group and HC group (both P<0.01), andwas significantly higher in EA group and NEA+NNA group than in HC group(both P<0.01), but there was no significant difference between EA group andNEA+NNA group (P>0.05).4. The level of IL-17in sputum supernatant was significantly higher in NA group than in EA group, NEA+NNA group and HCgroup (both P<0.01),and was significantly higher in EA group and NEA+NNAgroup than in HC group (both P<0.01), but there was no significant differencebetween EA group and NEA+NNA group (P>0.05). The level of IL-5in sputumsupernatant was significantly higher in EA group than in NA group, NEA+NNAgroup and HC group (both P<0.01), and was significantly higher in NA groupand NEA+NNA group than in HC group (both P<0.01), but there was nosignificant difference between NA group and NEA+NNA group (P>0.05). Thelevel of IL-8in sputum supernatant was significantly higher in NA group than inEA group, NEA+NNA group and HC group (bothP<0.01), and was significantlyhigher in EA group and NEA+NNA group than in HC group (both P<0.01), butthere was no significant difference between EA group and NEA+NNA group(P>0.05).5.The level of IL-17in plasma was no significant difference amongNA group,EA group,NEA+NNA group and HC group (P>0.05). The level ofIL-17in the culture supernatant from PMA-stimulated PBMC was significantlyhigher in NA group than in EA group, NEA+NNA group and HC group (bothP<0.01), and was significantly higher in EA group and NEA+NNA group than inHC group (both P<0.01), but there was no significant difference between EAgroup and NEA+NNA group (P>0.05). The levels of IL-17in the culturesupernatant from PMA-stimulated PBMC were significantly higher than thosein plasma in NA group,EA group and NEA+NNA group (both P<0.01),but therewas no significant difference between plasma level of IL-17and PBMC culturesupernatant level of IL-17in HC group(P>0.05).6. The percentage of Th17cellsin peripheral blood, the levels of IL-17in sputum and PBMC culturesupernatant,and the level of IL-8in sputum supernatant were all correlatedpositively with the percentage of neutrophils in sputum, Both FEV1%pred and PEF%pred were correlated negatively with the percentage of neutrophils insputum. The percentage of Th2cells in peripheral blood and the level of IL-5insputum supernatant were correlated positively with the percentage ofeosinophils in sputumConclusions Both Th17cells and Th2cells are involved in thepathogenesis of asthma in children. Th17cells and IL-17may mediateneutrophilic airway inflammation in asthma，indicating an important role forTh17cells in childhood neutrophilic asthma. Th17cells may be maintain theirsurvival through activation of the JAK/STAT5signaling pathway. Part twoEffect of inhaled glucocorticosteroid on Th17cell-mediatedneutronphilic airway inflammation inchildhood neutrophilic asthmaObjective To explore the effect of inhaled glucocorticosteroid on Th17cell-mediated neutrophilic airway inflammation in childhood neutrophilicasthma.Methods Children with neutrophilic asthma from NA group (n=10) inthe part one inhaled glucocorticosteroid over three months,and then underwentlung function tests and sputum induction. Sputum was assessed differential forcell count.Th17cells in peripheral blood and Ki-67expressed in Th17cells weredetected by flow cytometry. Expression of RORγt-mRNA in PBMC wasdetected by Real-time PCR. Concentrations of IL-17in induced sputum supernatant and in the culture supernatant from PMA-stimulated PBMC weremeasured by ELISA.Results1. FEV1%pred and PEF%pred were significantly higer aftertreatment than those before treatment in neutrophilic asthma(both p <0.01),butwere lower than those of HC group (p<0.05, p<0.01, respectively).2. Thepercentage of neutrophil in induced sputum was significantly lower aftertreatment than that before treatment in neutrophilic asthma (p<0.01), but washiger than that of HC group (p<0.05).3. The percentage of Th17cellsin peripheral blood was significantly lower after treatment than that beforetreatment in neutrophilic asthma (p<0.01), but was higer than that of HC group(p <0.01). The expression level of Ki-67in Th17cells was significantly lowerafter treatment than that before treatment in neutrophilic asthma (p<0.01), butwas higer than that of HC group (p<0.01).4. The expression level ofRORγt-mRNA in PBMC was significantly lower after treatment than thatbefore treatment in neutrophilic asthma (p<0.01), was no significant differentfrom that of HC group (P>0.05).5. The levels of IL-17in sputum and PBMCculture supernatant were significantly lower after treatment than those beforetreatment in neutrophilic asthma (both p<0.01), but were higer than those of HCgroup (p<0.05, p <0.01,respectively).Conclusions Inhaled glucocorticosteroid may reduce Th17cell-mediatedneutrophilic airway inflammation in childhood neutrophilic asthma bydecreasing percentage of Th17cells, sputum level of IL-17and down-regulatingRORγt expression.更多还原