TLR介导的ATP释放在副猪嗜血杆菌感染上调IL-1β中的作用研究

Haemophilus parasuis (HPS), one of the important bacterial diseases of swine industry in recent years, could cause serious pig lung inflammation. The experiment in vivo and vitro showed that the content of HPS is higher in lungs, NF-κB pathway could be activated，and the expression of IL-1β in pig alveolar macrophages increased significantly. At present, researchers indicated that TLR activation and the release of dangerous signals (ATP) in the inflammatory response played an important role in inflammation. This study will analyze the the mechanism of ATP release in IL-1β up-regulation by HPS with adding apyrase or ectogenic ATP, and study the influence of TLR activation to ATP release by using TLR4 inhibitor. In addition, the effect of P2X7 receptor and NLRP3 in IL-1βsecretion also will be analyzed by Real-time PCR、ELISA、Western-blot、TEM and laser confocal microscopy, which can clarify systematically the mechanism of TLR triggered ATP release in IL-1β up-regulation by HPS. The study could provide a new train of thought for the immune mechanism of HPS and the treatment of the disease , which has an important theoretical significance.

副猪嗜血杆菌（HPS）是近年来严重影响养猪业的重要细菌性疾病之一，能够引起猪肺部严重的炎症反应。我们前期通过动物攻毒试验发现肺脏HPS含菌量较高；细胞试验发现HPS能够激活猪肺泡巨噬细胞的NF-κB信号通路，并引起IL-1β表达量的显著上调。目前研究认为TLR的活化和危险信号ATP的释放在炎症反应中发挥重要作用。本研究拟将内源性ATP降解或添加外源性ATP来研究ATP释放在HPS感染上调IL-1β中的作用；使用TLR4抑制剂研究TLR的活化对ATP释放的影响；运用Real-time PCR、ELISA、Western-blot、激光共聚焦、TEM试验等技术方法，分析P2X7受体和NLRP3在IL-1β分泌过程中的作用，系统阐明TLR介导的ATP释放在HPS感染上调IL-1β中的作用机制。本研究可以为HPS的免疫机理及该病的治疗提供新的思路，具有重要的理论意义。

副猪嗜血杆菌病是近年来严重影响养猪业的重要细菌性疾病之一，能够引起猪肺部严重的炎症反应，目前研究认为TLR的活化和危险信号ATP的释放在炎症反应中发挥重要作用。我们通过热酚水法提取获得了高纯度的副猪嗜血杆菌脂多糖，明确了副猪嗜血杆菌及其脂多糖刺激猪肺泡巨噬细胞后，不同时间点均能引起细胞水平ATP释放量增加，胞外ATP的添加能够促进副猪嗜血杆菌感染的猪肺泡巨噬细胞中IL-1β的表达和分泌，且ATP能够影响猪肺泡巨噬细胞捕获、吞噬副猪嗜血杆菌的能力；通过沉默TLR4发现TLR4能够激活副猪嗜血杆菌脂多糖刺激猪肺泡巨噬细胞NF-κB信号通路，而且TLR4能够增强IL-1β的分泌和表达，该过程需要ATP/P2X7R介导的信号通路的参与和NLRP3炎性小体的活化。本课题系统阐明了TLR4介导的ATP释放在副猪嗜血杆菌及其脂多糖刺激猪肺泡巨噬细胞上调IL-1β分泌中的作用机制，为副猪嗜血杆菌病的免疫机理及该病的治疗提供新的思路，具有重要的理论意义。