肥大细胞对口蹄疫病毒样颗粒的模式识别作用与应答机制研究

Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a acute infectious disease of cloven-hoofed animals, threatening animal agriculture and food safety worldwide. At present, no successful control strategy has been officially approved. FMDV virus-like particles (VLPs) are nonreplicating, nonpathogenic particles in vivo that have structural characteristics and antigenicity similar to the parental virus, which may have potential as a new candidate vaccine antigen. Mast cells play pivotal roles both in innate immunity and adaptive immunity. However, the potential roles of mast cells in the immune responses to FMDV remain to be elusive. To this end, TLRs, scavenger receptor and mannose receptor expressed on mast cells are blocked with inhibitors. These mast cells are pulsed with FMDV VLP, the surrogate of native FMDV in vitro. Degranulation of mast cells are determined. Expression of iNOS and cytokines in supernatants is detected. DNA binding activity of NF-κB is detected. Distribution of mast cells and DC in mice subcutaneously immunized with FMDV VLP is microscopically observed. Simultaneously, the levels of antibody and cytokines in murine serum are detected in order to elucidate the mechanisms of pattern recognition of mast cells to FMDV VLP and the relevant biological outcome. It is believed that pending results of my winning proposal will pave the way foe novel vaccine development, thus, being of great theoretical significance and an appreciated expectation in practice.

口蹄疫（FMD）是由口蹄疫病毒（FMDV）感染偶蹄动物所引起的一种急性传染病，严重影响着全球畜牧业与食品安全。目前尚无有效的预防措施。FMDV病毒样颗粒（VLPs）是在体内不能复制、没有致病性的粒子，与亲本病毒有相似的结构特征和抗原特性，可能成为新的候选疫苗抗原。肥大细胞是天然免疫的效应细胞，并在适应性免疫应答过程中发挥启动与调节作用，但目前尚不清楚肥大细胞在机体抗FMDV免疫应答中的作用。本项目以FMDV VLP为材料，用相应的抑制剂阻断肥大细胞TLRs、清道夫受体和甘露糖受体，之后负载FMDV VLP，检测肥大细胞脱颗粒、iNOS、细胞因子的表达、NF- κB的DNA结合活性；用FMDV VLP 免疫小鼠，检测组织中肥大细胞及DC的分布、血清中抗体及细胞因子的水平，以阐明肥大细胞对FMDV的模式识别机制与生物学效应，为研制FMD新型疫苗提供科学依据，具有重要的理论意义和良好的应用前景。

口蹄疫（FMD）是由口蹄疫病毒（FMDV）感染偶蹄动物所引起的一种急性传染病，严重影响着全球畜牧业与食品安全。FMDV病毒样颗粒（VLP）是在体内不能复制、没有致病性的粒子，与亲本病毒有相似的结构特征和抗原特性，是一种理想的疫苗抗原形式。肥大细胞是天然免疫细胞，并在适应性免疫应答过程中发挥启动与调节作用。为了研究肥大细胞在机体抗FMDV免疫应答中的作用，本项目成功制备了VLP，用相应的抑制剂阻断小鼠腹腔肥大细胞（PMC）TLR2、TLR4、清道夫受体和甘露糖受体之后负载 VLP，发现PMC识别VLP所引发的脱颗粒主要是由TLR2和SR介导的。PMC通过TLR2识别VLP可诱导TNF-α分泌，但抑制IL-6分泌；通过TLR4识别VLP可引起TNF-α和IL-10分泌；但通过MR识别VLP则可抑制TNF-α、IL-6和IL-10分泌；值得注意的是，PMC通过SR识别VLP可引起促炎因子TNF-α和IL-6的分泌。利用MAPK和NF-κB的抑制剂处理PMC，然后负载VLP，结果显示PMC识别VLP 主要通过MAPK途径分泌TNF-α，而通过MAPK途径和NF-κB途径分泌IL-6，但IL-10的分泌与MAPK途径和NF-κB途径无关。再次给小鼠皮下注射VLP后，血清IgG水平显著高于初次免疫应答，表明VLP可以诱导B细胞免疫记忆。同时，血清TNF-α升高，但IL-10无明显变化。上述结果提示肥大细胞TLR2、TLR4、清道夫受体和甘露糖受体在识别VLP过程中发挥重要作用，这为深入研究VLP刺激肥大细胞应答的机制奠定了重要基础，并为VLP 疫苗佐剂的研制指明了方向。