诺如病毒通过HMGB1-RAGE axis引起肠道炎症反应的机制研究

Recently, acute gastroenteritis with highly contagious caused by norovirus break out frequently. NV infection cause severe vomiting and diarrhea, which is becomes the leading cause of gastroenteritis. However, the specific mechanism of NV infection is still unclear, and there is currently no vaccine or targeted drugs. Recently, we have found plenty of HMGB1 in the excrement from NV infection individuals. Pre-experiment showed that, in the Caco-2 cells, HMGB1 has transfer to the cytoplasm obviously, when stimulated the cells with NV virus-like particle. And also, the expression of RAGE (one of the receptor of HMGB1) was remarkable up-regulated in the biopsy samples of NV infection individuals. So, we speculate that NV could stimulate the enterocyte to release HMGB1 and subsequently activate the inflammatory reaction though HMGB1-RAGE axis. In this study, we intends to analysis the main source of HMGB1 that caused by NV infection. Explore and validate the mechanism that NV infection caused intestinal inflammatory reaction through HMGB1-RAGE axis. Furthermore, we will explore the roles of G82S in RAGE that contribute to change the inflammation levels through HMGB1-RAGE axis. This study will lay good foundation for further understanding the mechanism of NV infection，provide a good theoretical basis for target site selection in clinical intervention and individualized treatment.

近年来，由诺如病毒(NV)引起的急性肠胃炎爆发日益频繁且传染性极强，NV感染引发严重的呕吐与腹泻，已成为引起肠胃炎的主要原因。NV感染引发肠胃炎的具体机理尚不明确，目前亦无疫苗或针对性药物。最近，我们在NV感染患者排泄物中发现大量HMGB1，预实验发现受NV病毒样粒子刺激的结肠癌细胞核内HMGB1有明显向细胞质转移现象，而其受体基因RAGE在NV感染患者中有显著上调表达。我们推测NV感染可能通过刺激肠上皮细胞释放HMGB1并通过HMGB1-RAGE axis通路激活下游炎症反应。本研究拟探明NV感染后肠道内大量HMGB1的主要来源，探索并验证NV感染通过HMGB1-RAGE axis通路介导炎症反应的过程机制，并进一步分析RAGE基因G82S位点对该通路的影响。本项目为进一步理解NV感染的具体机理奠定良好的基础，同时为NV感染临床干预靶位点选择以及个体化治疗提供良好的理论依据。

本项目主要内容是研究诺如病毒（NV）通过RAGE-HMGB1通路引发肠道炎症反应的过程机制。在临床病理组织发现NV感染患者HMGB1水平显著升高。我们推测NV感染可能通过刺激肠上皮细胞释放HMGB1并通过HMGB1-RAGE axis通路激活下游炎症反应。首先，我们构建NV病毒外壳蛋白表达系统，成功获取病毒样粒子（VLP）。然后利用病毒VLP刺激肠道上皮细胞后，经过PCR检测发现细胞内HMGB1基因的mRNA水平显著上调；同时肠道HMGB1水平也显著上升。其次，通过QPCR检测发现HMGB1的受体基因RAGE也在VLP刺激的细胞内高表达，并且下游炎症因子也显著上调表达；而HMGB1的另外两个已知受体TLR2和TLR4表达水平没有显著变化。同时，我们WB结果也支持上述结果。此外，我们借助二代测序以及高分辨率熔解曲线技术筛选基因多态性通过病例对照试验发现RAGE以及HMGB1基因多态性位点可能会影响HMGB1-RAGE通路的信号传递引起肠道炎症风险差异。我们另外发现RAGE基因启动子区存在CpG甲基化岛，该区域甲基化成都也可能参与RAGE基因转录表达的调控。综上所述，我们围绕NV通过RAGE-HMGB1通路激发肠道炎症这一科学假说，验证了该假说的科学性，并探索了影响该通路的多种因素。最终得出初步结论认为NV在肠道中确实通过RAGE-HMGB1通路刺激下游炎症因子表达引起炎症反应，并且RAGE和HMGB1基因多态性以及RAGE启动子区甲基化水平均对该通路具有影响作用。该研究有利于进一步理解NV病毒感染引起炎症反应的机制，也为个性化诊断该通路相关病变提供理论依据。