H9N2猪流感病毒感染诱导细胞自噬的机制及其与NF-κB信号通路关系的研究

The present researching results have shown that the release of inflammatory cytokines is one of the most important pathogenic mechanisms of influenza virus, and cytokines were generated by the induction of autophagy. Our previous studies found that reactive oxygen species (ROS) was produced in the process of H9N2 swine influenza virus infection, the release of ROS is beneficial to the replication of the virus, and ROS can induce autophagy. So we speculated that the autophagy play an important role in influenza virus infection. To clarify the mechanism of autophagy induced by H9N2 swine influenza virus, first of all, A549 cell autophagy induced by the H9N2 swine influenza virus was observed，then the relationship between autophagy and viral replication was researched, the viral protein affected autophagy was searched, and the signal transduction pathways involved in autophagy was explored. Our preliminary results showed that the influenza virus infection was mediated by the nf-kappaB signaling pathway, but the concrete mechanism remains unclear. The objective of the research was to clarify the regulatory mechanism. And on this basis the relationship between the autophagy and nf-kappa B signaling pathway was explored during H9N2 swine influenza virus infection. It is the first time to link the autophagy and nf-kappa B signaling pathway during H9N2 swine influenza virus infection, the relevant report of those has not seen in domestic and foreign. The results of this study will provide the mechanism of H9N2 swine influenza virus infection in a new angle, which will provide new ideas in the prevention and treatment of the swine influenza or other influenza.

流感病毒的致病机制目前认为主要由于炎性细胞因子大量释放所致，而细胞自噬可以诱导细胞因子的产生。我们前期研究发现H9N2猪流感病毒感染可以产生活性氧，而活性氧释放促进病毒复制，并且活性氧可以诱导自噬。因此我们推测细胞自噬在流感病毒感染中发挥重要作用。本课题首先观察H9N2猪流感病毒感染诱导A549细胞自噬的发生，同时研究自噬与病毒复制的关系，寻找影响自噬的病毒蛋白，并探讨参与自噬发生的信号通路，旨在阐明H9N2猪流感病毒诱导自噬的机制。我们前期研究表明NF-κB通路介导了流感病毒的感染过程，但具体机制不详，本研究旨在探明其参与机制，在此基础上深入探讨H9N2猪流感病毒感染中NF-κB通路与细胞自噬的关系。本课题首次将H9N2猪流感病毒感染诱导细胞自噬与NF-κB通路联系起来，国内外尚未类似报道。研究结果将从新的视角探讨H9N2猪流感病毒致病机制，为猪流感和其它流感有效防治提供新思路和新靶点。

流感病毒感染主要呈现上呼吸道感染的急性传染性疾病, 部分感染者甚至发生严重肺炎，进而导致呼吸衰竭及多器官损伤，引起死亡。就其机制，目前研究认为由于炎性细胞因子大量释放所致，而自噬可以诱导细胞因子的产生。除此之外，研究发现H9N2猪流感病毒感染可以产生ROS，ROS可以诱导自噬。因此推测细胞自噬在H9N2猪流感病毒感染发挥重要的作用，基于此，本项目提出细胞自噬参与H9N2猪流感病毒感染的研究。如果研究证明细胞自噬参与H9N2猪流感病毒的感染，将为流感病毒的防控提供新的作用靶位和理论依据。.本研究进行了如下内容的研究，并取得了相应的成果：.1.H9N2猪流感病毒感染诱导A549细胞自噬：结果显示H9N2感染A549细胞后，透射电镜观察到自噬溶酶体，激光共聚焦显示细胞内的绿色荧光呈点状分布，WB证实LC3-II的表达量升高，说明该病毒可以诱导A549细胞自噬。.2.H9N2猪流感病毒编码蛋白对细胞自噬的影响：激光共聚焦显示转染M蛋白可以导致自噬的发生，而转染其他蛋白并未明显自噬现象。.3.H9N2猪流感病毒复制与细胞自噬：流感病毒感染导致自噬的发生，使用自噬抑制剂3-MA和LY294002以及Atg5 siRNA抑制细胞自噬,可降低病毒的滴度，降低NP mRNA 水平，降低NP和M蛋白的表达水平，说明抑制自噬可以抑制病毒的复制。 .4.H9N2猪流感病毒感染诱导A549细胞自噬与mTOR信号通路：mTOR信号通路参与H9N2诱导的细胞自噬。.5.H9N2猪流感病毒感染诱导A549细胞自噬与ERK1/2/mTOR信号通路：ERK、Akt/mTOR信号通路参与H9N2诱导的细胞自噬,抑制细胞自噬可以恢复由流感病毒引起的ERK、Akt/mTOR信号通路的改变情况。.6.H9N2猪流感病毒感染中NF-κB信号途径参与机制的研究：NF-κB信号通路通过经典激活途径参与H9N2诱导的自噬。.7.H9N2流感病毒感染中NF-κB信号通路与细胞自噬的关系：抑制自噬降低由流感病毒引起的p38、JNK、ERK以及NF-κB p65的表达上调以及mTOR的表达下调。抑制NF-κB信号通路，对自噬无影响。说明抑制自噬可以抑制NF-κB信号通路的激活，反之无影响。.本研究证实自噬在H9N2病毒的感染中发挥重要作用，这将为流感病毒的防控提供新的作用靶位和理论依据，具有重要的科学意义。