白细胞介素-25在哮喘中通过诱导nuocytes促进Th2型细胞因子表达的研究

发布时间：2017-12-27 13:08

本文关键词：白细胞介素-25在哮喘中通过诱导nuocytes促进Th2型细胞因子表达的研究　出处：《山东大学》2016年博士论文　论文类型：学位论文

【摘要】：支气管哮喘(asthma)是由多种炎症细胞如嗜酸性粒细胞(Eosinophils,EOS).肥大细胞、T淋巴细胞等及多种细胞组分参与的气道慢性炎症性疾病,并且通常以气道上皮的结构改变为病理特征。支气管哮喘的三个主要病理改变包括：气道炎症、气道重塑及平滑肌功能紊乱。慢性炎症反应体现在细胞因子和细胞介质的释放,这些炎症细胞因子和炎症介质可通过一定的途径引起哮喘气道炎性改变。哮喘使小气道痉挛和黏液大量分泌,引起气道狭窄和气道高反应。支气管哮喘的病因及发病机制复杂,目前尚未完全明确,现今研究较多围绕Th1/Th2免疫反应失衡学说。Th1/Th2平衡消长是机体免疫反应调节的基本方式,正常情况下,两者处于一种平衡状态,Th1/Th2细胞之间处于相互抑制的状态,而遗传变异及环境因素的变化可使两者的平衡优势改变,从而导致Th1/Th2比例关系趋向于Th2占优势,Th2相关的免疫反应功能亢进以及细胞因子分泌过多。CD4+Th2细胞活化释放白细胞介素-5(Interleukin,IL.5)口白细胞介素-13(Interleukin,IL-13)等炎症因子,促进嗜酸性粒细胞向气道募集、黏液过多分泌、上皮细胞增生肥大,促使B细胞转换为IgE分泌型细胞,导致血清中IgE水平增加,引起气道高反应性。多项研究显示,白细胞介素-25(Interleukin,IL.25)诱导Th2类型细胞因子如IL-5、IL-13产生,参与哮喘气道炎症的多个环节,并参与支气管哮喘引起的气道重塑,与支气管哮喘关系密切。研究还指出,固有免疫细胞也可生成IL-5和IL-13等Th2型细胞因子,因此推测在固有免疫细胞在Th2型免疫应答的启动中可能发挥着重要作用.Nuocytes是目前发现的Ⅱ型固有淋巴细胞(Type II innate lymphoid cells,ILC2s)之一,最早在肠道中发现,广泛存在于骨髓、外周血、脾脏中,但数量较少。目前越来越多的研究证明固有免疫细胞在细胞免疫应答中起重要作用。近年来发现nuocytes在细胞免疫应答中具有促进Th2型细胞因子表达的作用,在哮喘动物实验中也发现nuocytes的存在,并发现该细胞表面IL-25受体高表达,故nuocytes是否参与哮喘疾病中与Th2型细胞因子的形成及与IL-25的相互作用,成为哮喘疾病发生机制的研究热点,也为哮喘治疗开辟了新的方向。[研究目的]探讨支气管哮喘中IL-25及Th2型细胞因子表达的关系及其在哮喘气道炎症中的作用；并探讨IL-25在支气管哮喘中是否通过诱导nuocytes促进Th2型细胞因子表达。[研究方法]将BALB/C小鼠随机分为3组,分别命名为哮喘组、抗IL-25组和对照组,其中,给予哮喘组及抗IL-25组小鼠致敏后雾化吸入1%卵清蛋白(Ovalbumin, OVA)建造动物哮喘模型实验组,对照组小鼠用同体积0.9%氯化钠溶液雾化。另外,抗IL-25组经鼻滴入抗IL-25抗体(0.5mg/只),哮喘组及对照组经鼻注入等体积的0.9%氯化钠溶液。根据小鼠临床症状及肺组织病理变化等为标准判断造模是否成功。取各组小鼠的肺泡灌洗液,用于分析各组小鼠肺泡灌洗液中的白细胞及嗜酸粒细胞计数及嗜酸粒细胞占白细胞总数的比例；解剖各组小鼠获取肺组织标本,切片固定后采用HE染色观察各组小鼠肺组织病理学变化,应用酶联免疫反应(ELISA)、Western blot及实时定量-PCR(RT-PCR)等实验方法与技术测定哮喘组、抗IL-25组和对照组各组肺泡灌洗液、肺组织中IL-25及IL-5、IL-13等Th2型细胞因子的表达情况；运用流式细胞仪测定哮喘组、抗IL-25组和对照组三组小鼠肺泡灌洗液中nuocytes的数量。采用SPSS 17.0统计软件包进行统计学数据处理,所有数据均以均数和标准差标示,计量资料用t检验,检验水准a=0.05,P0.05为有统计学意义。分析IL-25及Th2型细胞因子表达的关系及其在哮喘气道炎症中的作用；并探讨IL-25在支气管哮喘中是否通过诱导nuocytes促进Th2型细胞因子表达。[结果](1)各组间肺泡灌洗液白细胞、嗜酸性粒细胞计数及嗜酸粒细胞比例：哮喘组小鼠肺泡灌洗液中白细胞总数、嗜酸性粒细胞绝对值及嗜酸性粒细胞百分比均较抗IL-25组和对照组小鼠明显增高,差别有统计学意义(P0.05)；而抗IL-25组小鼠肺泡灌洗液中白细胞总数、嗜酸性细胞绝对值及嗜酸性粒细胞百分比与对照组小鼠相比无明显差异(P0.05)。(2)HE染色哮喘组小鼠支气管粘膜下和肺泡周围募集大量炎症细胞浸润,黏液分泌细胞增多,支气管中有大量分泌物聚集；抗IL-25组小鼠整个肺组织及支气管周围均有少量细胞核成蓝染的炎性细胞聚集,少量的黏液分泌细胞增生,支气管中有少量分泌物聚集,呈轻度炎性病变的表现；对照组小鼠肺组织标本可见支气管粘膜下和肺泡周围无明显的炎症细胞浸润及分泌物。(3)通过酶联免疫吸附试验(ELISA)发现：哮喘组小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表达比抗IL-25组和对照组小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表达明显增高,有统计学意义(P0.05)；而抗IL-25组小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表达比与对照组小鼠肺泡灌洗液中IL-25、IL-5、IL-13的表达相比无明显差异(P0.05)。(4)通过WesternBlot及RT-PCR检测发现：哮喘组小鼠肺组织中IL-25蛋白及mRNA的表达比抗IL-25组和对照组小鼠肺组织中IL-25蛋白及mRNA的表达明显增强,具有统计学意义(P0.05)；而抗IL-25组小鼠IL-25蛋白及mRNA的表达与对照组小鼠肺组织中IL-25蛋白及mRNA的表达水平相比,两组间无明显统计学差异(P0.05)。(5)流式细胞术检测发现：哮喘组小鼠肺泡灌洗液中nuocytes的数量明显增多,与抗IL-25组和对照组小鼠肺泡灌洗液中nuocytes的数量相比,有统计学意义(P0.05)；而抗IL-25组小鼠肺泡灌洗液中nuocytes的数量与对照组小鼠肺泡灌洗液中nuocytes的数量表达增多,但无统计学差异(P0.05)。[结论]支气管哮喘模型中,支气管粘膜下和肺泡周围募集大量炎症细胞浸润,黏液分泌细胞增多。肺泡灌洗液中白细胞总数及嗜酸性粒细胞数量增加,支气管中有大量分泌物聚集,哮喘小鼠肺泡灌洗液及肺组织中IL-25及Th2型炎性细胞因子IL-5、IL-13表达增多。哮喘小鼠肺泡灌洗液中固有免疫细胞nuocytes明显增加,nuocytes表面可表达IL-25受体,应用抗IL-25抗体干预后小鼠肺泡灌洗液中nuocytes数量及IL-5、IL-13等相关的炎症指标明显减少,表明IL-25在哮喘中可通过诱导nuocytes促进Th2型细胞因子表达,抗IL-25抗体可阻断这一途径,有望成为哮喘治疗的新靶点。
[Abstract]:Bronchial asthma (asthma) is a chronic inflammatory disease involving many inflammatory cells, such as Eosinophils, EOS, mast cells, T lymphocytes and many other cellular components. Three major pathological changes in bronchial asthma include airway inflammation, airway remodeling, and smooth muscle dysfunction. Chronic inflammatory reaction is reflected in the release of cytokines and cell mediators. These inflammatory cytokines and inflammatory mediators can lead to airway inflammatory changes through a certain way. Asthma makes small airway spasms and mucus secrete, causing airway stenosis and airway hyperresponsiveness. The etiology and pathogenesis of bronchial asthma is complex, and it is not completely clear at present. Nowadays, many studies have focused on the theory of Th1/Th2 immune response imbalance. Th1/Th2 balance is the basic method to regulate the body's immune response, under normal circumstances, both in a balanced state, the mutual inhibition between Th1/Th2 cells in the state, and changes in genetic variation and environmental factors to balance the advantages of both changed, which causes the Th1/ Th2 ratio tends to the dominance of Th2, Th2 related immune the reaction function hyperfunction and cytokine hypersecretion. CD4+Th2 cell activation and release of interleukin -5 (Interleukin, IL.5) white interleukin -13 (Interleukin, IL-13) and other inflammatory cytokines, promote eosinophil recruitment to the airway, mucus hypersecretion, epithelial hyperplasia, prompting B cells into IgE secreting cells, leading to increased levels of IgE in serum. Induced airway hyperresponsiveness. Many studies have shown that interleukin -25 (Interleukin, IL.25) induces Th2 type cytokines, such as IL-5 and IL-13, is involved in many links of airway inflammation in asthma, and is involved in airway remodeling induced by bronchial asthma, which is closely related to bronchial asthma. The study also pointed out that innate immune cells can also generate IL-5 IL-13 and Th2 type cytokines, presumably in innate immune cells may play an important role in.Nuocytes is found in type II cells in the innate immune response priming in type Th2 (Type II innate lymphoid cells, ILC2s) one of the first found in the intestinal tract that widely exist in bone marrow, peripheral blood and spleen, but fewer. At present, more and more studies have shown that innate immune cells play an important role in cellular immune response. In recent years, found that nuocytes could promote the expression of Th2 type cytokines on cell immune response, nuocytes also found in asthma animal experiments, and found that the high expression of the cell surface receptor IL-25, the formation of the involvement of nuocytes in asthma and Th2 type cytokines and their interaction with IL-25, become a hot research topic the pathogenesis of asthma, also opened up a new direction for the treatment of asthma. [Objective] to explore the relationship between the expression of IL-25 and Th2 cytokines in bronchial asthma and its role in airway inflammation in asthma, and to explore whether IL-25 can induce nuocytes expression in bronchial asthma by promoting the expression of Th2 cytokines. [Methods] BALB/C mice were randomly divided into 3 groups, which were named as asthma group, anti IL-25 group and control group, the asthma group and anti IL-25 group treated mice were sensitized after inhalation of 1% ovalbumin (Ovalbumin, OVA) the construction of the experimental animal model of asthma group, control group of mice with the same volume of 0.9% Sodium Chloride Solution atomization. In addition, the anti IL-25 group was injected with anti IL-25 antibody (0.5mg/ only) through nasal drip, and 0.9% Sodium Chloride Solution in asthma group and control group were injected into the nose by nasal injection. The success of the model was judged according to the clinical symptoms and pathological changes of the lung tissue. Take the mice alveolar lavage, analysis of bronchoalveolar lavage fluid in leukocyte and eosinophil count and eosinophil leukocyte accounted for the proportion of the total used; anatomy of mice lung tissue were obtained after fixation by HE staining, sections of lung tissue were observed pathological changes, application of ELISA the reaction (ELISA), Western blot and real-time -PCR (RT-PCR) and other experimental methods and techniques for determination of anti asthma group, IL-25 group and control group bronchoalveolar lavage and lung tissue in IL-25 lotion, and IL-5, IL-13 and Th2 type cytokines expression; using flow cytometry, anti asthma group IL-25 three groups of mice in the bronchoalveolar lavage fluid nuocytes. SPSS 17 statistical software package was used for statistical data processing. All data were marked by mean and standard deviation. T was used to test data, and a=0.05 was tested. P0.05 was statistically significant. Objective to analyze the relationship between IL-25 and Th2 cytokines expression and its role in airway inflammation in asthma, and to explore whether IL-25 can induce nuocytes expression in bronchial asthma. [results] (1) between the groups in bronchoalveolar lavage fluid of white blood cells, eosinophil count and eosinophil percentage of asthmatic mice in BALF leukocyte count and eosinophil absolute value and percentage of eosinophils were anti IL-25 group and control group were significantly higher, statistically the significance of difference (P0.05); anti IL-25 mice bronchoalveolar leukocytes and eosinophils in BALF and the absolute value of the percentage of eosinophils compared with control mice showed no significant difference (P0.05). (2) HE staining around the asthmatic mice bronchial mucosa and alveolar recruitment inflammatory cell infiltration, mucus secretion of cells increased, a large number of bronchial secretions aggregation; anti IL-25 mice of the bronchus and lung tissue surrounding the nucleus into the blue has a small amount of inflammatory cell aggregation, a small amount of mucus secreting cells in bronchial hyperplasia. A small amount of secretions aggregation, mild inflammatory lesions; the control group around the mouse lung tissue specimen showed bronchial mucosa and alveolar no obvious inflammatory cell infiltration and
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R562.25

【相似文献】