IL-17A对哮喘气道嗜酸性粒细胞炎症的影响及其免疫机制研究
发布时间：2018-08-01 19:04
【摘要】：研究背景:支气管哮喘(以下简称“哮喘”)是一种具有明显异质性的慢性气道炎症性疾病。哮喘具有可逆性的气流受限、气道高反应性和慢性气道炎症等临床特征。根据哮喘的临床特征不同可以分为不同的临床表型,如过敏性和非过敏性哮喘等等。不同临床表型的哮喘其发病机制也有所不同,即存在“哮喘内型”差别。不同的内型可能解释不同哮喘“表型”的病理生理过程。目前发现内型为“Th2增高(Th2-high)”的患者,表现为过度增强的Th2型免疫应答,Th2细胞过度活化及其相关细胞因子IL-4、IL-5等的产生增多,同时气道以嗜酸性粒细胞浸润为主。临床上的过敏性哮喘为“Th2增高”的内型,通常由吸入的过敏原所诱发,具有明显的Th2型免疫反应,同时伴有气道内嗜酸性粒细胞、肥大细胞和淋巴细胞聚集,气道平滑肌的增生,以及血清Ig E的升高。IL-17A是一种主要由Th17细胞分泌的细胞因子,Th17细胞具有明显区别于Th1、Th2细胞的特征,其分化受到IFN-γ和IL-4的负性调控。IL-17A的主要生物学作用是在机体内通过引起中性粒细胞的聚集、活化,参与抵抗病原体的感染以及风湿类疾病等。IL-17A是参与中性粒细胞炎症非常重要的细胞因子。近年来研究发现哮喘患者的血和肺中IL-17A因子的浓度增高,并且和哮喘的严重程度相关,提示IL-17A可能参与了哮喘慢性气道炎症的发生和发展,特别是与中性粒细胞性哮喘有关。国内外多个研究中发现IL-17A具有促炎效应以及一定的抑炎效应,即IL-17A可能存在双重效应,研究中抑制炎症的效应可能是低浓度IL-17A所引起的。我们既往的研究发现,利用IL-17A基因敲除小鼠建立OVA诱导的哮喘模型同时给予LPS气道干预时较野生鼠的气道嗜酸性粒细胞炎症增高及脾脏Th2细胞分化增多。因此,我们假设IL-17A能通过直接抑制Th2细胞分化,减轻哮喘小鼠的气道嗜酸性粒细胞炎症。本研究通过OVA致敏和激发建立气道嗜酸性粒细胞浸润为主的哮喘小鼠模型,并观察IL-17A对哮喘小鼠气道嗜酸性粒细胞炎症以及Th2型免疫的影响及其机制。研究目的:1.探索IL-17A在哮喘小鼠气道炎症中的作用,包括肺部炎症细胞浸润程度以及BALF中细胞分类和细胞因子的变化。2.探索IL-17A在哮喘小鼠支气管淋巴结和脾脏以及体外培养中的Th2细胞分化中的作用。研究方法:第一章IL-17A对哮喘小鼠气道炎症的影响1.OVA诱导的哮喘小鼠模型的建立及气道干预处理实验分为三组,即对照组、模型组、IL-17A处理组。在建模第1、7天,模型组和IL-17A处理组小鼠腹腔注射OVA致敏,第14-18天用1%OVA雾化1h激发,对照组均予以等量生理盐水;IL-17A处理组小鼠每次激发前1h麻醉后气道滴入100 ng IL-17A,对照组及模型组予以等量生理盐水。2.BALF、肺组织标本的检测收集BALF液计细胞总数,离心后上清用ELISA法检测IL-4、IL-5、INF-γ、IL-17A浓度,所得沉淀涂片后染色行分类计数;左肺切片后行HE及PAS染色,于显微镜下观察进行半定量评分。第二章IL-17A对哮喘Th2细胞分化的抑制作用及机制1.小鼠支气管淋巴结及脾脏Th细胞分化的检测制备淋巴结和脾脏单细胞悬液,刺激培养后利用流式细胞术检测Th1/2/17的分化比例。2.IL-17A在体外诱导幼稚CD4+T细胞向Th2细胞分化中的抑制作用制备脾脏单细胞悬液,用免疫磁珠分选幼稚CD4+T细胞,用分化因子液加或者不加30 ng IL-17A同完全培养基一起培养细胞。在48h洗涤细胞后,用仅含IL-2的完全培养基继续培养至96h。利用流式细胞术检测培养24h、96h时Th细胞分化。利用Annexin V/PI法检测孵育24h时细胞凋亡程度。相同的培养条件下利用CCK8法比较培养24h时的增殖活力。研究结果:第一章IL-17A对哮喘小鼠气道炎症的影响1.BALF中细胞总数及各类细胞计数及其比例的比较模型组较对照组小鼠BALF中的细胞总数高,具有显著的统计学差异(P0.01),IL-17A处理组小鼠的BALF细胞总数明显低于模型组小鼠(P0.05);模型组BALF嗜酸性粒细胞数(P0.05)和比例(P0.01)明显高于对照组,而IL-17A处理组嗜酸性粒细胞数(P0.05)及其比例(P0.01)明显低于模型组,但IL-17A处理组嗜酸性粒细胞数及其比例仍高于对照组(P0.01);模型组及IL-17A处理组中性粒细胞数及淋巴细胞数较对照组有升高趋势,但差异无统计学意义(P0.05)。2.小鼠肺部支气管和血管周围炎症浸的比较对照组小鼠肺部未见有明显的炎症细胞浸润;模型组较对照组小鼠的支气管周围和血管周围有更明显的炎症细胞浸润,半定量评分更高(P0.01);予以IL-17A气道滴入后,IL-17A处理组小鼠支气管周围和血管周围炎症浸润程度明显低于模型组小鼠,且炎症评分有显著的统计学差异(P0.05);相比对照组,IL-17A处理组小鼠肺部的炎症浸润仍较显著(P0.01)。3.小鼠气道杯状细胞化生程度及半定量评分比较对照组小鼠的气道中未见明显的杯状细胞化生;模型组小鼠气道中较对照组可见更多的PAS阳性细胞,半定量评分具有显著差异(P0.01);IL-17A处理组较模型组小鼠的气道中杯状细胞化生程度低(P0.01)。4.小鼠BALF中Th相关细胞因子的比较模型组小鼠BALF上清中的Th1相关因子IFN-γ浓度显著低于对照组(P0.01),Th2相关因子IL-4、IL-5浓度显著高于对照组(P0.01),IL-17A浓度也显著高于对照组(P0.01);IL-17A处理组小鼠BALF中IL-4、IL-5浓度显著低于模型组(P0.01),IL-17A浓度显著高于模型组(P0.01),两组之间IFN-γ浓度无显著差异(P0.05);IL-17A处理组小鼠IL-4(P0.01)、IL-5(P0.05)浓度仍显著高于对照组,IFN-γ浓度显著低于对照组(P0.01),IL-17A浓度也显著高于对照组(P0.01)。第二章IL-17A对哮喘Th2细胞分化的抑制作用及机制1.小鼠支气管淋巴结中Th细胞分化的比较模型组小鼠淋巴结中Th1比例低于对照组(P0.01),Th2比例高于对照组(P0.05),两组之间Th17比例无显著性差异(P0.05);IL-17A处理组较模型组小鼠有更低的淋巴结Th2比例(P0.05),两组Th1和Th17比例之间差异无统计学意义(P0.05);IL-17A处理组Th2比例显著高于对照组小鼠(P0.05),Th1比例显著低于对照组小鼠(P0.01),两组小鼠Th17比例之间差异无统计学意义(P0.05)。2.小鼠脾脏Th细胞分化程度比较模型组小鼠脾脏Th1比例低于对照组(P0.01),Th2比例显著高于对照组(P0.01),两组Th17比例之间无显著差异(P0.05);给予IL-17A气道滴入后,IL-17A处理组小鼠脾脏Th2比例显著低于模型组(P0.01),两组之间Th1和Th17均无显著差异(P0.05);IL-17A处理组小鼠脾脏Th1细胞比例仍低于对照组(P0.01),两组之间Th17比例差异不显著(P0.05)。3.IL-17A在体外诱导幼稚CD4+T细胞向Th2细胞分化中的抑制作用在24h时,Th2极化组和Th2+IL-17A极化组之间细胞凋亡和增殖均无显著差异(P0.05)。Th2+IL-17A极化组中Th2细胞分化比例在培养24h时较Th2极化组呈下降趋势(P0.05),在培养至96h,IL-17A明显抑制Th2细胞分化(P0.01)。研究结论:1.给OVA复制的哮喘模型气道滴入IL-17A后可减轻肺部嗜酸性粒细胞浸润和降低BALF中Th2相关因子IL-4、IL-5的浓度。而且,不导致气道中性粒细胞炎症。2.哮喘小鼠中支气管淋巴结和脾脏的Th2细胞比例明显高于对照组,IL-17A气道滴入后可以降低Th2细胞分化比例,而不影响Th1细胞比例。IL-17A在体外还可抑制幼稚CD4+T细胞向Th2细胞分化,而对其增殖和凋亡无显著影响。研究意义:哮喘的“卫生学说”认为,儿童时期接触细菌内毒素等可以通过刺激Th1细胞分化,来抑制Th2的分化,改善Th2/Th1失衡,从而减少哮喘的发病。本文的研究证实了,通过气道内给予IL-17A处理,也可以抑制Th2的分化,改善哮喘气道的嗜酸细胞性炎症。因为细菌内毒素是刺激机体Th17分化的重要原因。因此,本文结果丰富了对“卫生学说”免疫调节机制的认识。我们实验室既往的研究显示,过强的内毒素刺激可以导致过强的Th17细胞反应,从而导致哮喘气道的嗜酸细胞性炎症转换为中性粒细胞炎症,给哮喘的治疗带来困难。但我们研究所有相对较低剂量的IL-17A处理哮喘模型,并没有导致明显的中性粒细胞炎症。
[Abstract]:Background: bronchial asthma (hereinafter referred to as "asthma") is a chronic airway inflammatory disease with distinct heterogeneity. Asthma has the clinical characteristics of reversible airflow limitation, airway hyperresponsiveness, and chronic airway inflammation. The clinical characteristics of asthma can be divided into different clinical phenotypes, such as anaphylaxis and anaphylaxis. Asthma and so on. The pathogenesis of asthma in different clinical phenotypes is also different, that is, the difference in the "type of asthma type". Different internal types may explain the pathophysiological process of different asthma "phenotypes". The patients with "Th2 increase (Th2-high") "are currently found to be over enhanced Th2 type immune response and Th2 cell overactivity. The production of IL-4, IL-5, and other related cytokines are increased and the airway is mainly eosinophil infiltration. The clinical allergic asthma is the internal type of "Th2 increase", usually induced by inhaled allergens, with an obvious Th2 type immune response, accompanied by the accumulation of eosinophils, mast cells and lymphocytes in the airway. The proliferation of airway smooth muscle and the increase of Ig E in serum.IL-17A is a cytokine secreted mainly by Th17 cells. Th17 cells have distinct characteristics that are distinct from Th1, Th2 cells, and their differentiation by IFN- gamma and IL-4's negative regulation of.IL-17A is the main biological function of neutrophils in the body by causing aggregation and activation of neutrophils. .IL-17A, which is involved in resistance to pathogens and rheumatic diseases, is a very important cytokine involved in the inflammation of neutrophils. In recent years, it has been found that the concentration of IL-17A in the blood and lungs of the asthmatic patients is higher and is associated with the severity of asthma, suggesting that IL-17A may be involved in the occurrence and development of chronic airway inflammation in asthma. The development of IL-17A is particularly associated with neutrophil asthma. Many studies have found that IL-17A has a proinflammatory effect and a certain anti inflammatory effect, that is, IL-17A may have double effects. The effect of inhibition of inflammation in the study may be caused by low concentration of IL-17A. Our previous study found that the use of IL-17A gene knockout mice to establish OVA The induced asthma model also gave LPS airway intervention to increase airway eosinophil inflammation and splenic Th2 cell differentiation in the wild rats. Therefore, we hypothesized that IL-17A could reduce airway eosinophil inflammation in asthmatic mice by directly inhibiting the differentiation of Th2 cells. This study established airway eosinophilia through OVA sensitization and stimulation. The effect of IL-17A on airway eosinophil inflammation and Th2 type immunity in asthmatic mice and its mechanism were observed. 1. the purpose of this study was to explore the role of IL-17A in airway inflammation in asthmatic mice, including the degree of infiltration of inflammatory cells in the lungs, and the changes in cell classification and cytokine in BALF. 2. to explore the role of IL-17A in the differentiation of Th2 cells in bronchial lymph nodes, spleen and in vitro culture of asthmatic mice. Research methods: the first chapter: the effect of IL-17A on the airway inflammation in asthmatic mice, the establishment of 1.OVA induced asthma mice model and the airway intervention treatment were divided into three groups: the control group, the model group and the IL-17A treatment group. On day 1,7, the mice in the model group and the IL-17A treatment group were intraperitoneally sensitized by OVA, and 1H was stimulated with 1%OVA atomization on day 14-18. The control group was equal to the same amount of physiological saline; the airway was dropped into 100 ng IL-17A before each time of 1H anesthesia in the IL-17A treatment group and the control group and the model group were equal to the same amount of normal saline.2.BALF, and the detection collection of lung tissue specimens was collected BALF. The total number of cells in the liquid meter, after centrifugation, the concentration of IL-4, IL-5, INF- gamma, IL-17A was detected by ELISA method. After the precipitation smear, the staining was classified. The left lung slices were stained with HE and PAS, and the semi quantitative score was observed under the microscope. Second chapter IL-17A on the differentiation of asthma Th2 cells and mechanism 1. mice bronchial lymph node and spleen T. H cell differentiation was used to prepare the lymphoid and splenic single cell suspension. After stimulation, the differentiation ratio of Th1/2/17 was detected by flow cytometry,.2.IL-17A was used to induce the splenic single cell suspension in vitro induced by the inhibition of the immature CD4+T cells to the Th2 cell differentiation. The immunomagnetic beads were used to separate the infantile CD4+T cells, and the differentiation factor solution was added to the cell suspension. The cells were cultured together with the complete medium without 30 ng IL-17A. After 48h scrubbing cells, a complete medium containing only IL-2 was used to continue to be cultured to 96h. by flow cytometry to detect the differentiation of Th cells when 24h and 96h were cultured. Annexin V/PI method was used to detect the degree of apoptosis when incubating 24h. The effect of IL-17A on airway inflammation in asthmatic mice: the total number of cells and the number of cells in 1.BALF and the proportion of cells in the model group were higher than those in the control group of BALF, and there were significant statistical differences (P0.01). The total number of BALF cells in the IL-17A group was significantly lower than that of the model group. The BALF eosinophil number (P0.05) and proportion (P0.01) in the model group were significantly higher than that in the control group, but the eosinophil number (P0.05) and its proportion (P0.01) in the IL-17A treatment group were significantly lower than that in the model group, but the eosinophil number and the proportion of the IL-17A treated group were still higher than that of the control group (P0.01); the number of neutrophils in the model group and the IL-17A treatment group was more than that of the control group (P0.01). The number of eosinophils and the proportion of the IL-17A treatment group were still higher than the control group (P0.01). The number of neutrophils in the model group and the IL-17A treatment group was more than that of the control group (P0.01). There was no significant difference in the number of lymphocytes in the control group, but the difference was not statistically significant (P0.05). There was no obvious inflammatory cell infiltration in the lungs of the bronchi and blood vessels around the lung of the.2. mice. The model group had more obvious inflammatory cell infiltration around the bronchi and around the blood tube in the control group than the control group. The score was higher (P0.01). After IL-17A airway drip, the inflammatory infiltration around and around the bronchi in IL-17A treatment group was significantly lower than that in the model group, and the inflammatory score was significantly different (P0.05). Compared with the control group, the inflammatory infiltration in the lung of the IL-17A treatment group was still more significant (P0.01).3. mouse airway goblet cells No obvious goblet cell metaplasia was found in the airway of the control group. More PAS positive cells were found in the model group than the control group, and the semi quantitative score was significantly different (P0.01) in the model group. The IL-17A treatment group was lower than the goblet cell metaplasia in the model mice (P0.01).4. mice BALF The concentration of Th1 related factors IFN- gamma in BALF supernatant was significantly lower than that of control group (P0.01), and the concentration of Th2 related factor IL-4, IL-5 concentration was significantly higher than that of control group (P0.01), and IL-17A concentration was significantly higher than that of control group (P0.01), and the concentration of Th1 was significantly lower than that of the model group. The concentration of A was significantly higher than that of the model group (P0.01), and there was no significant difference in IFN- gamma concentration between the two groups (P0.05). The concentration of IL-4 (P0.01) and IL-5 (P0.05) in the IL-17A treatment group was still significantly higher than that of the control group. The concentration of IFN- gamma was significantly lower than the control group (P0.01), and the IL-17A concentration was significantly higher than that of the control group. The inhibitory effect of the second chapter on the differentiation of asthma cells was significantly higher than that of the control group. The proportion of Th1 in the lymph node of 1. mice was lower than that of the control group (P0.01), and the proportion of Th2 was higher than that of the control group (P0.05). There was no significant difference in the proportion of Th17 between the two groups (P0.05), and the IL-17A treatment group had lower lymph node Th2 ratio (P0.05), and the two groups of Th1 and Th17 ratios were between the two groups. The difference was not statistically significant (P0.05), the proportion of Th2 in the IL-17A treatment group was significantly higher than that of the control group (P0.05), and the proportion of Th1 was significantly lower than that of the control group (P0.01). There was no significant difference in the proportion of Th17 in the two groups (P0.05) the Th1 proportion of the spleen Th cells in the spleen of the mice was lower than that of the control group (P0.01), and the proportion of the spleen was lower than the control group. Significantly higher than the control group (P0.01), there was no significant difference in the proportion of Th17 between the two groups (P0.05). After the infusion of IL-17A airway, the proportion of Th2 in the spleen of the IL-17A treatment group was significantly lower than that in the model group (P0.01), and there was no significant difference between the Th1 and Th17 (P0.05) between the two groups (P0.05), and the proportion of the splenic Th1 cells in the IL-17A treatment group was still lower than that of the control group, between the two groups. The inhibition effect of.3.IL-17A on the differentiation of CD4+T cells to Th2 cells in vitro was not significant (P0.05). There was no significant difference in the cell apoptosis and proliferation between the Th2 polarization group and the Th2+IL-17A polarization group at 24h (P0.05) in the.Th2+IL-17A polarization group, the percentage of Th2 cells in the.Th2+IL-17A polarization group was lower than that in the Th2 polarization group. IL-17A significantly inhibited Th2 cell differentiation (P0.01) in the culture of 96h. Conclusions: 1. after IL-17A in the airway of the OVA replicated asthma model, the infiltration of eosinophils and the decrease of Th2 related factors IL-4, IL-5 concentration in BALF are reduced, and the bronchial lymph nodes and spleen in the asthmatic mice of airway neutrophilic granulocytic inflammation are not caused. The proportion of Th2 cells was significantly higher than that in the control group. The percentage of Th2 cell differentiation could be reduced after IL-17A airway drip, without affecting the proportion.IL-17A of Th1 cells to inhibit the differentiation of infant CD4+T cells to Th2 cells in vitro, but there was no significant effect on its proliferation and apoptosis. Endotoxin and so on can stimulate the differentiation of Th1 cells to inhibit the differentiation of Th2, improve the Th2/Th1 imbalance, and reduce the incidence of asthma. This study confirmed that IL-17A treatment in the airway can also inhibit the differentiation of Th2 and improve the eosinophilic inflammation in the airway of asthma, because bacterial endotoxin is the heavy stimulation of the body's Th17 differentiation. The results of this paper enrich the understanding of the immunomodulatory mechanism of the "health doctrine". Previous studies in our laboratory have shown that excessive endotoxin stimulation can lead to excessive Th17 cell responses, leading to the conversion of eosinophilic inflammation in the airway to neutrophil inflammation, but it is difficult for the treatment of asthma. We studied all the relatively low doses of IL-17A in the treatment of asthma models without causing significant neutrophil inflammation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25
本文编号：2158538
[Abstract]:Background: bronchial asthma (hereinafter referred to as "asthma") is a chronic airway inflammatory disease with distinct heterogeneity. Asthma has the clinical characteristics of reversible airflow limitation, airway hyperresponsiveness, and chronic airway inflammation. The clinical characteristics of asthma can be divided into different clinical phenotypes, such as anaphylaxis and anaphylaxis. Asthma and so on. The pathogenesis of asthma in different clinical phenotypes is also different, that is, the difference in the "type of asthma type". Different internal types may explain the pathophysiological process of different asthma "phenotypes". The patients with "Th2 increase (Th2-high") "are currently found to be over enhanced Th2 type immune response and Th2 cell overactivity. The production of IL-4, IL-5, and other related cytokines are increased and the airway is mainly eosinophil infiltration. The clinical allergic asthma is the internal type of "Th2 increase", usually induced by inhaled allergens, with an obvious Th2 type immune response, accompanied by the accumulation of eosinophils, mast cells and lymphocytes in the airway. The proliferation of airway smooth muscle and the increase of Ig E in serum.IL-17A is a cytokine secreted mainly by Th17 cells. Th17 cells have distinct characteristics that are distinct from Th1, Th2 cells, and their differentiation by IFN- gamma and IL-4's negative regulation of.IL-17A is the main biological function of neutrophils in the body by causing aggregation and activation of neutrophils. .IL-17A, which is involved in resistance to pathogens and rheumatic diseases, is a very important cytokine involved in the inflammation of neutrophils. In recent years, it has been found that the concentration of IL-17A in the blood and lungs of the asthmatic patients is higher and is associated with the severity of asthma, suggesting that IL-17A may be involved in the occurrence and development of chronic airway inflammation in asthma. The development of IL-17A is particularly associated with neutrophil asthma. Many studies have found that IL-17A has a proinflammatory effect and a certain anti inflammatory effect, that is, IL-17A may have double effects. The effect of inhibition of inflammation in the study may be caused by low concentration of IL-17A. Our previous study found that the use of IL-17A gene knockout mice to establish OVA The induced asthma model also gave LPS airway intervention to increase airway eosinophil inflammation and splenic Th2 cell differentiation in the wild rats. Therefore, we hypothesized that IL-17A could reduce airway eosinophil inflammation in asthmatic mice by directly inhibiting the differentiation of Th2 cells. This study established airway eosinophilia through OVA sensitization and stimulation. The effect of IL-17A on airway eosinophil inflammation and Th2 type immunity in asthmatic mice and its mechanism were observed. 1. the purpose of this study was to explore the role of IL-17A in airway inflammation in asthmatic mice, including the degree of infiltration of inflammatory cells in the lungs, and the changes in cell classification and cytokine in BALF. 2. to explore the role of IL-17A in the differentiation of Th2 cells in bronchial lymph nodes, spleen and in vitro culture of asthmatic mice. Research methods: the first chapter: the effect of IL-17A on the airway inflammation in asthmatic mice, the establishment of 1.OVA induced asthma mice model and the airway intervention treatment were divided into three groups: the control group, the model group and the IL-17A treatment group. On day 1,7, the mice in the model group and the IL-17A treatment group were intraperitoneally sensitized by OVA, and 1H was stimulated with 1%OVA atomization on day 14-18. The control group was equal to the same amount of physiological saline; the airway was dropped into 100 ng IL-17A before each time of 1H anesthesia in the IL-17A treatment group and the control group and the model group were equal to the same amount of normal saline.2.BALF, and the detection collection of lung tissue specimens was collected BALF. The total number of cells in the liquid meter, after centrifugation, the concentration of IL-4, IL-5, INF- gamma, IL-17A was detected by ELISA method. After the precipitation smear, the staining was classified. The left lung slices were stained with HE and PAS, and the semi quantitative score was observed under the microscope. Second chapter IL-17A on the differentiation of asthma Th2 cells and mechanism 1. mice bronchial lymph node and spleen T. H cell differentiation was used to prepare the lymphoid and splenic single cell suspension. After stimulation, the differentiation ratio of Th1/2/17 was detected by flow cytometry,.2.IL-17A was used to induce the splenic single cell suspension in vitro induced by the inhibition of the immature CD4+T cells to the Th2 cell differentiation. The immunomagnetic beads were used to separate the infantile CD4+T cells, and the differentiation factor solution was added to the cell suspension. The cells were cultured together with the complete medium without 30 ng IL-17A. After 48h scrubbing cells, a complete medium containing only IL-2 was used to continue to be cultured to 96h. by flow cytometry to detect the differentiation of Th cells when 24h and 96h were cultured. Annexin V/PI method was used to detect the degree of apoptosis when incubating 24h. The effect of IL-17A on airway inflammation in asthmatic mice: the total number of cells and the number of cells in 1.BALF and the proportion of cells in the model group were higher than those in the control group of BALF, and there were significant statistical differences (P0.01). The total number of BALF cells in the IL-17A group was significantly lower than that of the model group. The BALF eosinophil number (P0.05) and proportion (P0.01) in the model group were significantly higher than that in the control group, but the eosinophil number (P0.05) and its proportion (P0.01) in the IL-17A treatment group were significantly lower than that in the model group, but the eosinophil number and the proportion of the IL-17A treated group were still higher than that of the control group (P0.01); the number of neutrophils in the model group and the IL-17A treatment group was more than that of the control group (P0.01). The number of eosinophils and the proportion of the IL-17A treatment group were still higher than the control group (P0.01). The number of neutrophils in the model group and the IL-17A treatment group was more than that of the control group (P0.01). There was no significant difference in the number of lymphocytes in the control group, but the difference was not statistically significant (P0.05). There was no obvious inflammatory cell infiltration in the lungs of the bronchi and blood vessels around the lung of the.2. mice. The model group had more obvious inflammatory cell infiltration around the bronchi and around the blood tube in the control group than the control group. The score was higher (P0.01). After IL-17A airway drip, the inflammatory infiltration around and around the bronchi in IL-17A treatment group was significantly lower than that in the model group, and the inflammatory score was significantly different (P0.05). Compared with the control group, the inflammatory infiltration in the lung of the IL-17A treatment group was still more significant (P0.01).3. mouse airway goblet cells No obvious goblet cell metaplasia was found in the airway of the control group. More PAS positive cells were found in the model group than the control group, and the semi quantitative score was significantly different (P0.01) in the model group. The IL-17A treatment group was lower than the goblet cell metaplasia in the model mice (P0.01).4. mice BALF The concentration of Th1 related factors IFN- gamma in BALF supernatant was significantly lower than that of control group (P0.01), and the concentration of Th2 related factor IL-4, IL-5 concentration was significantly higher than that of control group (P0.01), and IL-17A concentration was significantly higher than that of control group (P0.01), and the concentration of Th1 was significantly lower than that of the model group. The concentration of A was significantly higher than that of the model group (P0.01), and there was no significant difference in IFN- gamma concentration between the two groups (P0.05). The concentration of IL-4 (P0.01) and IL-5 (P0.05) in the IL-17A treatment group was still significantly higher than that of the control group. The concentration of IFN- gamma was significantly lower than the control group (P0.01), and the IL-17A concentration was significantly higher than that of the control group. The inhibitory effect of the second chapter on the differentiation of asthma cells was significantly higher than that of the control group. The proportion of Th1 in the lymph node of 1. mice was lower than that of the control group (P0.01), and the proportion of Th2 was higher than that of the control group (P0.05). There was no significant difference in the proportion of Th17 between the two groups (P0.05), and the IL-17A treatment group had lower lymph node Th2 ratio (P0.05), and the two groups of Th1 and Th17 ratios were between the two groups. The difference was not statistically significant (P0.05), the proportion of Th2 in the IL-17A treatment group was significantly higher than that of the control group (P0.05), and the proportion of Th1 was significantly lower than that of the control group (P0.01). There was no significant difference in the proportion of Th17 in the two groups (P0.05) the Th1 proportion of the spleen Th cells in the spleen of the mice was lower than that of the control group (P0.01), and the proportion of the spleen was lower than the control group. Significantly higher than the control group (P0.01), there was no significant difference in the proportion of Th17 between the two groups (P0.05). After the infusion of IL-17A airway, the proportion of Th2 in the spleen of the IL-17A treatment group was significantly lower than that in the model group (P0.01), and there was no significant difference between the Th1 and Th17 (P0.05) between the two groups (P0.05), and the proportion of the splenic Th1 cells in the IL-17A treatment group was still lower than that of the control group, between the two groups. The inhibition effect of.3.IL-17A on the differentiation of CD4+T cells to Th2 cells in vitro was not significant (P0.05). There was no significant difference in the cell apoptosis and proliferation between the Th2 polarization group and the Th2+IL-17A polarization group at 24h (P0.05) in the.Th2+IL-17A polarization group, the percentage of Th2 cells in the.Th2+IL-17A polarization group was lower than that in the Th2 polarization group. IL-17A significantly inhibited Th2 cell differentiation (P0.01) in the culture of 96h. Conclusions: 1. after IL-17A in the airway of the OVA replicated asthma model, the infiltration of eosinophils and the decrease of Th2 related factors IL-4, IL-5 concentration in BALF are reduced, and the bronchial lymph nodes and spleen in the asthmatic mice of airway neutrophilic granulocytic inflammation are not caused. The proportion of Th2 cells was significantly higher than that in the control group. The percentage of Th2 cell differentiation could be reduced after IL-17A airway drip, without affecting the proportion.IL-17A of Th1 cells to inhibit the differentiation of infant CD4+T cells to Th2 cells in vitro, but there was no significant effect on its proliferation and apoptosis. Endotoxin and so on can stimulate the differentiation of Th1 cells to inhibit the differentiation of Th2, improve the Th2/Th1 imbalance, and reduce the incidence of asthma. This study confirmed that IL-17A treatment in the airway can also inhibit the differentiation of Th2 and improve the eosinophilic inflammation in the airway of asthma, because bacterial endotoxin is the heavy stimulation of the body's Th17 differentiation. The results of this paper enrich the understanding of the immunomodulatory mechanism of the "health doctrine". Previous studies in our laboratory have shown that excessive endotoxin stimulation can lead to excessive Th17 cell responses, leading to the conversion of eosinophilic inflammation in the airway to neutrophil inflammation, but it is difficult for the treatment of asthma. We studied all the relatively low doses of IL-17A in the treatment of asthma models without causing significant neutrophil inflammation.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R562.25
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