自噬在病毒感染加速COPD发病进程中的作用及其相关机制研究

发布时间：2018-03-06 07:28

本文选题：COPD　切入点：自噬　出处：《浙江大学》2015年博士论文　论文类型：学位论文

【摘要】：COPD是全世界范围内发病率和死亡率最高的疾病之一,是一种常见的以持续性气流受限为特征的可以预防和治疗的疾病。吸入香烟烟雾和其他有毒颗粒,如生物燃料的烟雾导致的肺脏炎症是COPD发生的重要原因,其中吸烟是世界范围内引起COPD最常见的危险因素。此外,在许多国家,大气污染、职业暴露、室内生物燃料污染也是引起COPD的主要危险因素。当今,COPD的发病机制尚未完全清楚。目前普遍认为COPD与单核/巨噬细胞、中性粒细胞、T淋巴细胞引起的炎症反应、蛋白酶与抗蛋白酶失衡以及氧化应激反应等相关。微生物感染是COPD急性加重的重要原因,但有研究指出,在COPD小气道病变的发生中,细菌感染可能没有直接作用。另有研究发现COPD患者都存在隐性的或潜在的腺病毒感染。COPD患者肺和气道内皮细胞中均有腺病毒转录激活蛋白的表达,而且与香烟引起的炎症应答的扩大化密切相关。提示腺病毒感染可能是COPD急性发作的病因之一。近年来,随着研究者们对COPD发病机制不断深入的研究,一个新发现的程序性细胞死亡类型的生物过程受到了人们的关注——自噬。越来越多的研究发现COPD的发生发展与自噬有着密切的关系,不论是在COPD患者还是烟雾暴露的小鼠中都发现了增强的自噬水平。那么,自噬在COPD的发生发展中具有怎样的作用?在腺病毒引起COPD加重的过程中自噬又起到了怎样的作用呢? 我们以COPD小鼠模型、小鼠气道上皮细胞和小鼠3LL细胞系为研究对象,结合体外细胞共培养等实验技术以期阐述自噬在COPD进展及腺病毒加重COPD进展过程中的作用。本研究发现：1、以香烟提取物(cigarette smoking extract, CSE)处理小鼠气道上皮细胞、3LL细胞系后细胞中LC3B表达量增加,p62表达量减少,自噬的发生水平增高,提示CSE能够诱导细胞发生自噬；2、以CSE处理3LL细胞系后,再以腺病毒进行感染,与单纯CSE处理的细胞相比,细胞中LC3B表达量更多,p62表达量更少,自噬发生水平更高,提示腺病毒感染能够加速CSE诱导的自噬发生进程；3、3LL细胞系以CSE处理后,再以腺病毒感染,继之与小鼠CD8+T细胞共培养,CD8+T细胞IFN-γ的分泌量及穿孔素的释放量在CSE或CSE与病毒的刺激下有所增加,而在经过自噬抑制剂预处理后降低,提示腺病毒感染可通过自噬途径来诱导CD8+T细胞功能的活化；4、经过长期香烟烟雾暴露的COPD模型小鼠肺组织中CD8+T细胞数量增加,且IFN-y的分泌量及穿孔素的释放量也具有上升趋势,证明CD8+T细胞在肺部的募集及功能的活化导致了COPD的发生发展过程中肺组织的损伤,而肺组织中LC3B的表达量增加,p62表达量减少,自噬水平升高,进一步证明自噬对CD8+T细胞在肺部募集及功能活化的促进作用。
[Abstract]:COPD is one of the highest morbidity and mortality rates in the world and is a common preventable and treatable disease characterized by persistent airflow limitation. For example, inflammation of the lungs caused by the smoke of biofuels is an important cause of COPD, among which smoking is the most common risk factor for COPD worldwide. In addition, in many countries, air pollution, occupational exposure, Indoor biofuel pollution is also the main risk factor of COPD. The pathogenesis of COPD is not completely clear. It is generally believed that COPD and mononuclear / macrophage, neutrophil T lymphocytes cause inflammatory reaction. Protease is associated with antiproteinase imbalance and oxidative stress reaction. Microbial infection is an important cause of acute exacerbation of COPD, but some studies have shown that in the pathogenesis of COPD small airway disease, Bacterial infection may not play a direct role. Other studies have found that adenovirus transcriptional activators are expressed in both lung and airway endothelial cells in patients with COPD. These results suggest that adenovirus infection may be one of the causes of acute attack of COPD. In recent years, with the further study of the pathogenesis of COPD, A newly discovered biological process of programmed cell death has attracted much attention-autophagy. More and more studies have found that the development of COPD is closely related to autophagy. Enhanced autophagy levels have been found in both COPD patients and smog exposed mice. So what is the role of autophagy in the development of COPD? What role does autophagy play in the process of COPD aggravation caused by adenovirus? We studied COPD mouse model, mouse airway epithelial cells and mouse 3LL cell lines. The purpose of this study was to elucidate the role of autophagy in the progression of COPD and the aggravation of COPD by adenovirus in combination with in vitro cell coculture. In this study, we found that the mouse airway epithelial cell line 3LL was treated with cigarette extract smoking extract (CSE1). The expression of LC3B increased and the expression of p62 decreased. The increased level of autophagy suggested that CSE could induce autophagy 2, then infect 3LL cell line with CSE, and then infect with adenovirus. Compared with the cells treated with CSE alone, the expression of LC3B in the cells was less than that in the cells treated with CSE. The level of autophagy was higher, which suggested that adenovirus infection could accelerate the process of autophagy induced by CSE and then infect with adenovirus after treated with CSE. Then, the secretion of IFN- 纬 and the release of perforin of CD8 T cells co-cultured with mouse CD8 T cells increased under the stimulation of CSE or CSE and virus, but decreased after pretreatment with autophagy inhibitor. The results suggested that adenovirus infection could induce the activation of CD8 T cells by autophagy pathway, and the number of CD8 T cells in lung tissues of COPD model mice exposed to cigarette smoke for a long time increased. The secretion of IFN-y and the release of perforin also showed an increasing trend, which proved that the recruitment and activation of CD8 T cells in the lung resulted in lung injury during the development of COPD. However, the expression of LC3B increased and the expression of p62 decreased, while the level of autophagy increased, which further demonstrated the role of autophagy in promoting the recruitment and functional activation of CD8 T cells in the lungs.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R563.9

【共引文献】