新型葡萄球菌肠毒素引发猪多系统炎症的分子机理

Severve pathological lesion and diversified inflammation reactions can be caused by Staphlococcal enterotoxion，it interrogate a global health and food safety challenge owing to a widely Staphloccus aureus infection.It is still unclear about the molecular mechanism and pathogenesis about some noval Staphlococcal enterotoxion superantigens. .The molecular mechanism of pathogenesis,involved in molecular structure of six enterotoxins，the immunological synapse between the surface molecules of T lymphocytes, APC cells and their immune response caused by six recombinant Staphlococal enterotoxin superantigen will be illustrated by developing two models (coculture of antigen-present cells and T lymphocytes model, swine infection model).The structure epitope of enterotoxin will be analyzed by constructing enterotoxin mutatants,pepscan ELISA detection,and monoclonal antibody to block the specific binding epitope of enterotoxins; The immunological synapase formed by TCR,MHC II and enterotoxin will be explored by real-time RT-PCR, confocal microscopy,flow cytometry and proteomics 2D electrophrosis anylsis techniques.The immune response and changes caused by different entertoxin stmulation in APC & T cells,including co-stimulate molecules expression,the transcription and expression of cytokines will be detected; Intracellular signals produced in macrophages (phosphorylated MyD88, MyD88 downstream proteins IRAK4 and TRAF6) & TCR pathway signals (P-Lck、LAT、ZAP70、PLCΥ1) produced by T lymphocytes will be compared to staphlococcal enterotoxins unstimulated cells. The results of intestinal tract permeability and immune respone in swine experiment will be combined with the data of cell co-culture method to show the molecular mechanism and pathogenesis of staplococcal enterotoxin infection.The anticipated result should be helpful to understand the enterotoxin superantigen mechanism.

葡萄球菌肠毒素能引起多组织炎性损伤。本研究拟以6种重组肠毒素为研究对象，从肠毒素分子结构、细胞受体及其作用细胞的应答反应来认识新型肠毒素超抗原作用机理及对猪多系统炎症的影响。应用肠毒素重组突变体及单抗、Pepscan、单抗阻滞试验分析肠毒素的结构表位及其功能；APC、T细胞共培养体系探讨肠毒素与靶细胞表面互作分子的结构特点；应用荧光定量PCR、激光共聚焦显微技术、流式细胞术等检测APC、T细胞表面共刺激分子、细胞因子、信号转导分子的转录、表达变化；评价猪APC、T细胞对肠毒素的体内应答效应。预期可明确肠毒素超抗原与T、APC靶细胞表面分子作用的免疫突触单元，认识新型肠毒素超抗原活性及其在猪多系统炎症中的作用，丰富对葡萄球菌肠毒素的理论及应用研究。

在前期获得6种新型肠毒素蛋白（SEA、SEO、SEQ、SEK、SEG、SEU）的基础上，研究了其超抗原活性及其在引起猪多系统炎症的机制。.聚焦主要免疫突触形成分子的结构及功能研究，同源模建分析了6种葡萄球菌肠毒素与猪MHC II分子的互作界面氢键作用力，从强到弱为：SEO>SEQ>SEK> SEA>SEU>SEG。肠毒素α3-β8环可选择性特异识别TCRVβ的CDR2、CDR3区空间结构，α3-β8环长度及氨基酸残基差异是造成6种肠毒素超抗原活性差异的主要原因。研究发现，（1）不同肠毒素结合MHCⅡ类分子、TCRVβ 的潜在位点存在差异，超抗原活性的发挥受猪APC上MHC、T细胞上TCRVβ的识别限制；（2）不同肠毒素选择性活化特定的TCRVβ亚群T细胞；（3）肠毒素可影响免疫突触辅助分子CD28/ CTLA的表达，从而调节炎症反应。除形成免疫突触中央单元的MHC II-TCRVβ分子，多种细胞表面免疫辅助分子（B7-CD28、CD86、CD25、ICAM-LFA-1，B7-CTLA）参与了免疫突触的形成调节。.体内外实验研究了肠毒素对肠道上皮细胞、APC和T细胞细胞表面、信号转导、细胞因子的转录表达的影响。肠毒素通过与APC细胞上MHC II分子、T细胞上TCRVβ形成免疫突触，活化多种受体信号，调节多种免疫分子及细胞因子的转录表达；促进免疫突触形成分子（MHC II、CD4、TCRVβ，B7-CD28、CD86、CD25、ICAM-LFA-1，B7-CTLA）、细胞趋化功能（多种炎性因子、趋化因子受体）及胞内信号转导分子的转录表达；调节猪肠道上皮细胞、巨噬细胞、PBMC等IL-1β、TNF-α、GM-CSF、MCP-1、MIP-1β的转录。体内外促进T细胞活化增殖，分泌IFN-γ、IL-2、IL-4增强，形成复杂的炎症因子风暴。.除超抗原途径活化T细胞外，肠毒素可通过活化TLR途径（上调猪TLR6）、调节FcR形成不同剪接体形成，导致更为复杂的炎性因子风暴产生反应。这丰富了对葡萄球菌肠毒素早期快速致炎机理的认识。