【作者】 宋爽；

【导师】 陈焕春；

【作者基本信息】 华中农业大学 ， 预防兽医学， 2013， 博士

【摘要】 猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的一种严重危害养猪业的病毒性传染病,主要表现为母猪严重的繁殖障碍及仔猪的呼吸系统疾病和生长受阻。PRRSV为单股正链RNA病毒,自20世纪80年代末爆发以来,PRRSV已经严重威胁到了全球养猪业的发展,并且造成了巨大的经济损失。自1995年我国报道发生该病以来,该病己成为困扰我国养猪生产中最重要的病毒性传染病之一。特别是2006年在我国出现的高致病性蓝耳病,导致全国各地大批生猪死亡,给我国养猪业造成了巨大的经济损失。PRRSV感染猪可以引起强烈的间质性肺炎,提示我们炎症反应在PRRSV感染和致病性方面扮演着十分重要的角色。然而,目前对这种毒株感染后引起宿主细胞的信号转导机制知之甚少。针对这种新的PRRSV,本文开展了对其诱导宿主细胞产生炎症反应信号转导机制的研究,为阐明PRRSV的致病机理以及其新型疫苗的研制提供理论依据。具体研究内容包括：1.高致病性PRRSV感染激活IL-1β的分子机制及其生物学意义的研究IL-1β是一种主要由单核细胞、巨噬细胞和淋巴细胞产生的促炎性细胞因子,是机体调节免疫和炎症反应的中心介质,能介导多种炎症反应,诱导致炎性细胞因子的基因表达及分泌,在炎性疾病的致病中扮演着十分重要的角色。以前的研究已经发现,PRRSV能够诱导高水平的IL-1β。但其诱导的分子机制仍未阐明。本研究利用PRRSV感染猪体内的宿主细胞-PAM细胞,发现PRRSV激活IL-1β mRNA的表达和蛋白分泌,同时也分析了几种模式识别受体及其接头分子,发现TLR4/MyD88信号级联及其下游的信号通路NF-κB, ERK1/2和p38MAPK均参与PRRSV激活IL-1β。通过siRNA干扰和特异性抑制剂发现NLRP3炎症小体参与PRRSV诱导IL-1p的分泌,但不影响其激活IL-1β mRNA水平的表达；能够诱导NLRP3炎症小体形成的ROS和心外流也参与PRRSV诱导IL-1β的分泌。我们同时还发现TLR4/MyD88/NF-KB信号通路参与PRRSV诱导NLRP3炎症小体组成元件的表达。此外,本研究还分析了PRRSV诱导IL-1β产生的生物学意义。通过IL-1β纯化蛋白及其中和性抗体处理PAM细胞后发现,IL-1β对PRRSV的增殖无明显影响,但其中和性抗体能够抑伟PRRSV激活其它的促炎性因子(IL-6,IL-8)的表达,说明IL-1β在PRRSV诱导炎症反应中处于主导地位。2.高致病性PRRSV感染激活IL-6的分子机制的研究IL-6是一种多效的细胞因子,调节多种生物进程,包括神经系统、造血系统、应激反应、炎症与免疫应答。通常情况下,连同与促炎性细胞因子TNF-αt和IL-1β一起产生。IL-6通常在机体急性反应的情况下被诱导,主要被用来维持细胞动态平衡,也参与调节炎症反应,主要是细胞因子和组织炎症反应。作为一种促炎性细胞因子,IL-6在病毒感染中扮演着十分重要的角色。PRRSV感染也能够激活IL-6的表达,但其具体分子机制仍不清楚。本研究发现,PRRSV能够诱导高水平的IL-6产生,无论是mRNA还是蛋白水平。同时还发现TLR4/MyD88信号级联及其下游信号通路NF-κB, ERK1/2和p38MAPK均参与PRRSV激活IL-6。通过siRNA干扰和特异性抑制剂发现NLRP3炎症小体参与PRRSV诱导IL-6的表达,同时ROS和疋外流也参与PRRSV诱导IL-6的分泌。阐明了PRRSV感染激活IL-6的分子机制。此外,我们还分析了IL-6对PRRSV增殖的影响,通过IL-6纯化蛋白处理PAM细胞后发现,IL-6对PRRSV的增殖无明显影响。3.高致病性PRRSV感染激活1I,-8的分子机制的研究IL-8,或称为CXCL8,是一种促炎性CXC家族的趋化因子。IL-8的产生主要受到NF-r.B调控,同时NF-IL-6转录结合位点也参与调控IL-8启动子活性。多种刺激例如炎症反应信号(IL-1p和TNF-a的产生)、化学和环境压力(化疗和缺氧)以及类固醇激素能够产生IL-8。IL-8作为一种促炎性趋化因子在炎症反应中扮演着十分重要的角色。PRRSV在不同组织均能诱导高水平的IL-8产生。通过PRRSV感染PAM细胞分析了PRRSV感染激活IL-8的分子机制。研究发现,PRRSV能够诱导高水平的IL-8产生,无论是mRNA还是蛋白水平。我们还发现TLR4/MyD88信号级联及其下游的信号通路NF-κB, ERK1/2和p38MAPK均参与PRRSV激活IL-8。通过siRNA干扰和特异性抑制剂发现NLRP3炎症小体参与PRRSV诱导IL-8的表达,同时ROS和K+外流也参与PRRSV诱导IL-8的分泌。同时还分析了IL-8对PRRSV增殖的影响,通过IL-8纯化蛋白处理PAM细胞后发现,IL-8对PRRSV的增殖无明显影响。更多还原

【Abstract】 Porcine reproductive and respiratory syndrome (PRRS) is a viral infection disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), has a serious hazard to the swine industry, mainly represents a serious sow reproductive failure and respiratory system disease and growth stagnatim of piglets. PRRSV is a single-stranded RNA virus, since the outbreak in the late1980s, PRRSV has been a serious threat to the global development of pig industry, and caused huge economic losses. Since China reported the disease at1995, it has become one of the most important viral diseases in swine production in China. Especially in China at2006, the appearance of highly pathogenic PRRS, resulting in a large number of pigs were killed across the country, causing huge economic losses to the swine industry in our country. PRRSV can cause a strong interstitial pneumonia in infected pigs, suggesting that the inflammatory response plays a very important role in PRRSV infection and pathogenicity, however, it is poorly understood. For this new PRRSV, we analyzed the signal transduction mechanism of inflammatory response in PRRSV-infected host cell to provide a theoretical basis in order to clarify the pathogenesis of PRRSV. The specific studies include:1. The molecular mechanism of highly pathogenic PRRSV infection activate IL-ip and its biological significanceIL-1β is a pro inflammatory cytokines primarily produced by monocytes, macrophages and lymphocytes, the center medium of body regulating immune and inflammatory responses, can mediate a variety of inflammatory reactions, induces other inflammatory cytokines expression and secretion, plays a very important role in the pathogenesis of inflammatory diseases. Previous studies have found out that PRRSV can induce high levels of IL-1β, but the molecular mechanisms are not yet elucidated. In this study, we used PAMs, the target cells of PRRSV-infected pigs, found PRRSV activates IL-1β both at mRNA expression and protein secretion level. We also analyzed several pattern recognition receptor and its adapter molecule, found out that TLR4/MyD88signal cascade and its downstream signaling pathways including NF-κB, ERK1/2and p38MAPK are involved in the the PRRSV activation of IL-1β. The siRNA interference and specific inhibitor treatment indicated that NLRP3inflammasome involved in PRRSV-induced IL-1β secretion, but didn’t affect the activation of the expression of IL-1β mRNA levels. We also found that the activator to induce NLRP3inflammasome formation-ROS and K+efflux is also involved in PRRSV-induced IL-1β secretion. We also found TLR4/MyD88/NF-KB signaling pathway involved in PRRSV-induced NLRP3inflammasome component expression. In addition, we also analyzed the biological significance of PRRSV-induced IL-1β. Treatment of purified IL-1β protein and its neutralizing antibody on PAMs had no significant effect on the proliferation of PRRSV, but IL-1β neutralizing antibodies can inhibit the the PRRSV activation of other proinflammatory (IL-6, IL-8), indicating the centrol role of IL-1β in PRRSV-induced inflammatory response.2. The molecular mechanism of highly pathogenic PRRSV infection activate IL-6IL-6is a pleiotropic cytokine regulating a variety of biological processes, including the nervous system, the hematopoietic system, stress response, inflammation and immune response. Usually, produced together with the pro-inflammatory cytokines TNF-a and IL-1, IL-6is usually induced in the case of the body in acute reaction, is primarily be used to maintain cell homeostasis, and is also involved in the regulation of inflammation, cytokines and tissue inflammation reaction. As a pro-inflammatory cytokines, IL-6plays a very important role in viral infection. PRRSV infection is also capable of activating the expression of IL-6, but its specific molecular mechanisms are still unclear. Our study found that, PRRSV can induce high levels of IL-6, either on the mRNA or protein levels. We also found that TLR4/MyD88signaling cascade and its downstream signaling pathway of NF-κB, ERK1/2and p38MAPK are involved in PRRSV-induced IL-6. The siRNA interference and specific inhibitor treatment indicated that NLRP3inflammasome involved in PRRSV-induced IL-6production. We also found that ROS and K+efflux is also involved in PRRSV-induced IL-6secretion. In addition, we also analyzed the impact of IL-6on PRRSV proliferation. Treatment of purified IL-6protein on PAMs had no significant effect on the proliferation of PRRSV.3. The molecular mechanism of highly pathogenic PRRSV infection activate IL-8IL-8, or referred to CXCL8, is a CXC family pro-inflammatory chemokine. IL-8production mainly regulated by NF-κB, meanwhile NF-IL-6transcription binding sites is also involved in the regulation of the activity of the IL-8promoter. A variety of stimuli such as inflammatory response signal (IL-1(3and TNF-a generation), chemical and environmental stress (chemotherapy and hypoxia) and steroids can produce IL-8. As a pro-inflammatory chemokine IL-8plays a very important role in the inflammatory response summary. PRRSV is able to induce high level of IL-8in different organization. We used the PAMs, the target cells of PRRSV-infected pigs, to analyze the molecular mechanism of PRRSV infection activats IL-8. Our study found that, PRRSV can induce high levels of IL-8, either the mRNA or protein levels. We also found that TLR4/MyD88 signaling cascade and its downstream signaling pathway of NF-κB, ERK1/2and p38MAPK are involved in PRRSV-induced IL-8. The siRNA interference and specific inhibitor treatment indicated that NLRP3inflammasome involved in PRRSV-induced IL-8production. We also found that ROS and K+efflux is also involved in PRRSV-induced IL-8secretion. In addition, we also analyzed the impact of IL-8on PRRSV proliferation. Treatment of purified IL-8protein on PAMs had no significant effect on the proliferation of PRRSV.更多还原