Th17细胞与儿童中性粒细胞性哮喘气道炎症相关性研究

发布时间：2018-05-08 08:32

本文选题：哮喘 + 中性粒细胞　；参考：《广西医科大学》2014年博士论文

【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(p0.01)，EA组、NEA+NNA组Th17细胞百分比较HC组增高(p0.01)，EA组与NEA+NNA组间Th17细胞百分比比较差异无统计学意义(p0.05)；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(p0.01)，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(p0.05)；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(p0.01)，EA组、NEA+NNA组Th17细胞Ki-67表达阳性率较HC组增高(p0.01)，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(p0.05)；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(p0.01)，EA组、NEA+NNA组Th17细胞STAT5表达阳性率较HC组增高(p0.01)，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(p0.05)；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(p0.01)，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(p0.05)；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(p0.01)，EA组、NEA+NNA组RORγt-mRNA表达较HC组增高(p0.01)，EA组与NEA+NNA组间RORγt-mRNA表达比较差异无统计学意义(p0.05)；4、NA组痰上清液IL-17浓度较EA组、NEA+NNA组、HC组均明显增高(p0.01)，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(p0.05)；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(p0.05)；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(p0.05)；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(p0.05)，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(p0.01)，EA组、NEA+NNA组PBMC培养上清液IL-17浓度较HC组增高(p0.01)，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(p0.05)；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(p0.05)。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(p0.01)，但仍高于健康对照组（P0.05）；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p0.01)，但仍高于健康对照组（均P0.01）；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(p0.01)，与健康对照组比较差异无统计学意义(p0.05)；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：p0.05，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。
[Abstract]:Bronchial asthma (asthma) is a chronic inflammatory disease in the airway, which is involved in a variety of inflammatory cells (eosinophils, mast cells, T lymphocytes, neutrophils, etc.), airway structural cells (airway epithelial cells, smooth muscle cells, fibroblasts) and a variety of cytokines. The pathogenesis of asthma is complex and has not yet been fully explained. Chronic airway inflammation is the essence of asthma. It is important to study the pathogenesis of airway inflammation in asthma. The development mechanism has important clinical guiding significance for the prevention and treatment of asthma. With the in-depth study of airway inflammation in asthma, people's understanding of asthma is no longer limited to eosinophil airway inflammation, but more emphasis is placed on the common participation of multiple cells in asthma. Chronic airway inflammation. The present study shows that there are different subtypes of airway inflammation in asthma, which can be divided into eosinophilicasthma (EA) and non-eosinophilic asthma (NEA), and more than half of the non eosinophilic asthma is neutrophils (NA), but asthma (neutrophilic asthma, NA). The mechanism of polymorphonuclear neutrophils has not yet been fully elucidated. Auxiliary T lymphocytes (T helper cell, Th cells) play an important immunomodulatory role in the chronic airway inflammation of asthma. The dominant Th1/Th2 cell imbalance in.Th2 cells is considered to be the immunological basis for the formation and development of airway eosinophilic inflammation in allergic asthma. However, some studies have found that patients with non allergic asthma are not dependent on Th2 cell immunological mechanism mediated airway neutrophil inflammation. In recent years, Th17 cells and their effector IL-17 have been found to play an important role in allergic diseases such as bronchial asthma. IL-17 has a strong recruitment, the role of activating neutrophils, and the possibility of activating neutrophils. It is one of the mechanisms leading to the increase of neutrophils in the airway. However, the role of Th17 cells in the pathogenesis of different airway inflammation is the same. It is rarely reported whether there is a differentiation advantage of Th17 cells in the pathogenesis of neutrophils. The results showed that there were strong Th17 cell immunity, moderate Th2 cell immune response, Th17 cells mediated the pathogenesis of neutrophil airway inflammation in asthmatic mice by IL-17, and the differentiation of Th17 cells by up regulation of the transcription factor ROR gamma T, and the differentiated Th17 cells in neutrophil asthmatic mice were in the machine. It is the first issue to study whether there is the same Th17 cell differentiation advantage in the pathogenesis of human neutrophil asthma under the high IL-7 environment, which depends on the activation of the JAK/STAT5 signal pathway.
Glucocorticoid as the most effective anti inflammatory drug for asthma is mainly to reduce airway inflammation by reducing the activation of T lymphocytes and inhibiting the production of cytokines. Standardized asthma therapy based on inhaled corticosteroid (ICS) for most asthmatic patients can achieve asthma control, but some patients still have no expectations. In recent years, some studies have found that glucocorticoids do not inhibit Th17 cells and their effects on airway neutrophil inflammation mediated by IL-17, but there are also a few studies. The study showed that glucocorticoid therapy could reduce the expression of IL-17. We also found that dexamethasone could reduce the expression of ROR gamma t in the Th17 cell specific transcription factor of neutrophils, reduce the number of Th17 cells and the related cytokines IL-17 level, and partly reduce the airway neutrality of asthmatic mice. But dexamethasone has no significant effect on the prolonged survival of Th17 cells and the rate of apoptosis in airway neutrophils. At present, there are different results on the role of glucocorticoid in the airway inflammation mediated by Th17 cells. We will explore the correlation between Th17 cells and airway inflammation in children with neutrophils. On the basis of this, the effect of glucocorticoid on airway inflammation in children with neutrophils induced by Th17 cells is further explored. This study will preliminarily clarify the role of Th17 cells in the pathogenesis of neutrophil asthma in children and provide a new clue and theoretical basis for the treatment of neutrophilic asthma in children and help to asthma. The individualized treatment of asthma is divided into two parts:
Part 1 Correlation Analysis of Th17 cells and related cytokines with airway inflammation in children with neutrophilic asthma
Objective to explore the correlation between Th17 cells and related cytokines and airway inflammation in children with neutrophilic asthma.
Methods 28 cases of acute exacerbation of asthma were randomly selected, and the pulmonary function was measured and the sputum cell classification was induced. According to the induced sputum cytology, 12 cases were divided into three groups: 12 cases of eosinophilic asthma (group EA), 10 cases of neutrophil asthma (group NA) and non eosinophilic non neutrophil asthma (NE In group A+NNA) 6 cases were selected and 10 cases of healthy control group (group HC) were selected. Flow cytometry was used to detect the expression of Ki-67, STAT5, BCL-2 on the peripheral blood Th17, Th2 and Th17 cells, and the real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) was used to detect the expression of ROR gamma t-mRNA in peripheral blood mononuclear cells. PBMC stimulated IL-17 concentration in supernatant, IL-8 and IL-5 concentration in sputum supernatant and correlation analysis.
Results 1, the percentage of Th17 cells in peripheral blood Th cells in group NA was significantly higher than that in group EA, NEA+NNA group and HC group (P0.01), EA group and NEA+NNA group Th17 cell percentage was higher than HC group (P0.01). Group HC was increased (P0.01), group NA and group NEA+NNA were higher than group HC (P 0.01), and there was no significant difference in the percentage of Th2 cells between NA and NEA+NNA groups (P0.05). 2. Compared with group HC (P0.01), there was no significant difference in the positive rate of Ki-67 expression in Th17 cells between group EA and NEA+NNA group (P0.05), and the positive rate of STAT5 expression of Th17 cell STAT5 in peripheral blood of NA group was higher than that in EA group. The positive rate of STAT5 expression was not statistically significant (P0.05), and the positive rate of BCL-2 expression in Th17 cells in group NA was significantly higher than that in group EA, NEA+NNA group and HC group (P0.01), and there was no significant difference between the EA group, NEA+NNA group and HC group. The expression of ROR y in group NEA+NNA and group HC increased significantly (P0.01), and the expression of ROR gamma t-mRNA in group EA and NEA+NNA was higher than that in group HC (P0.01), and there was no statistical difference between EA group and NEA+NNA group. 4. There was no significant difference in the concentration of IL-17 in the sputum supernatant between the EA group and the NEA+NNA group (P0.05). The concentration of IL-5 in the sputum supernatant in the EA group was higher than that in the NA group, the NEA+NNA group and the HC group increased (P 0.01). The concentration of the sputum supernatant was higher than that of the group HC (0.01). The concentration of the sputum supernatant was higher than that of the group (0.01). There was no statistical significance (P0.05), and the concentration of IL-8 in the sputum supernatant in group NA was higher than that in group EA, NEA+NNA and HC (P 0.01), EA and NEA+NNA group was higher than that of HC group in group NEA+NNA (P 0.01). 5 There was no statistical significance (P0.05), but after PMA stimulation, the concentration of IL-17 in PBMC culture supernatant of NA group was higher than that of EA group, NEA+NNA group and HC group increased significantly (P0.01), EA group and NEA+NNA group were higher than those in the group of PBMC culture supernatant. P0.05); at the same time, after PMA stimulation, the concentration of IL-17 in the PBMC culture supernatant of group NA, EA and NEA+NNA was significantly higher than that of plasma IL-17 (P 0.01). There was no significant difference between the IL-17 concentration of the PBMC culture supernatant and the plasma concentration in HC group. The proportion and the IL-17 concentration of the supernatant IL-17, IL-8, PBMC culture supernatant was positively correlated, the percentage of NEU in the induced sputum in children with asthma was negatively correlated with FEV1%pred and PEF%pred, and the percentage of EOS in the induced sputum in children with asthma was positively correlated with the proportion of Th2 cells in the peripheral Th cells and the IL-5 concentration of the sputum.
Conclusion 1, Th17 cells and Th2 cells are all involved in the pathogenesis of childhood asthma. The expression of Th17 cells in peripheral blood, the specific transcription factor ROR gamma T and the level of IL-17 in sputum in children with neutrophils are significantly higher than those of eosinophilic and non eosinophilic non neutrophilic granulocytic asthma, and it is also associated with the induction of sputum neutrophils. The proportion is positive correlation. It suggests that Th17 cells may mediate the occurrence of airway inflammation in neutrophil asthma by IL-17, and the survival of the differentiated Th17 cells in neutrophilic asthma in children may be prolonged, which may be related to the activation of the JAK/STAT5 signal pathway.
The second part is the effect of inhaled corticosteroids on airway inflammation in Th17 cell mediated neutrophilic asthma in children.
Objective to investigate the effects of inhaled corticosteroids on airway inflammation induced by Th17 cells in children with neutrophilic asthma.
Methods in the first part of the study, 10 children with neutrophils (NA) were studied. After 3 months of regular inhalation of glucocorticoid under the guidance of the physician, the lung function was measured and the phlegm cell classification was induced. Flow cytometry was used to detect the expression of Ki-67 on the peripheral blood and Th17 cells; real-time fluorescence quantitative polymerization was used. Real-time PCR was used to detect the expression of ROR t-mRNA in peripheral blood mononuclear cells. ELISA method was used to detect sputum supernatant and PBMC culture supernatant IL-17 concentration.
Results 1, FEV1%pred and PEF%pred were significantly higher in group NA after inhaling corticosteroid than before treatment (all P 0.01), but still lower than that in healthy control group (P 0.05); 2, group NA was significantly lower before treatment (P0.01) after inhaled corticosteroid therapy, but still higher than that in healthy control group (P0.05); 3, NA group rule inhalation. The expression of Ki-67 in peripheral blood Th17 cells and Th17 cells in peripheral blood was significantly lower than before treatment (all P0.01), but still higher than that in the healthy control group (all P0.01). 4, the expression of ROR y t-mRNA in peripheral blood mononuclear cells of group NA was significantly lower than that before treatment (P0.01), and there was no difference compared with that of the healthy control group. Study significance (P0.05); 5, the concentration of IL-17 in the sputum supernatant and PBMC culture supernatant after inhaled corticosteroid treatment in group NA was lower than that before treatment (P0.05, P 0.01), but still higher than that in the healthy control group (P 0.01).
Conclusion inhaled corticosteroids can reduce the level of expression of specific transcription factor ROR gamma T, to a certain extent, reduce the level of IL-17 in peripheral blood Th17 cells and sputum, and reduce airway inflammation in children with neutrophilic granulocytic asthma mediated by Th17 cells.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R725.6

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