鸭坦布苏病毒入侵血-脑屏障的细胞分子机制

作为首发于我国的重大畜禽疫病病原，鸭坦布苏病毒（DTMUV）的感染过程具有明显时序性。在揭示了DTMUV突破血-脾屏障（BSB）机理的基础上，课题组将从血-脑屏障（BBB）的新角度继续探究该病毒的致病机制。应用形态技术分析DTMUV引发BBB结构变化的细胞学特征，显示病毒在脑组织的分布定位，厘清内皮膜联蛋白A2、钙黏蛋白与各种紧密连接蛋白的表达动态，阐释感染鸭BBB血管连接复合体损伤与屏障通透性改变的内在规律。进一步应用免疫共沉淀、激光共聚焦和免疫胶体金技术，深入研究DTMUV感染期A2、钙黏蛋白以及连接相关蛋白的互作关系，解析病毒下调A2表达、降低钙黏蛋白动员与紧密连接蛋白再组装的分子机理。同时，静脉注射外源重组A2，验证DTMUV入侵对BBB屏障作用的影响机制。研究结果将与上个项目的实验发现一起，完整、系统地阐明DTMUV的致病机理，为DTMUV病的防治与疫苗开发提供有效的理论支撑。

As a pathogen of major livestock and poultry disease,which was firstly found in China,Duck Tembusu virus (DTMUV) infects ducks with temporal sequence.After the implementation of dynamic analysis on DTMUV breakthrough the blood-spleen barrier (BSB) by our group,the infection mechanism of DTMUV is further revealed from the new perspective of blood-brain barrier (BBB) in this project.The cellular pathological changes of infected duck brain and its BBB are observed by using morphological techniques.The virus distribution in brain is showed by immunofluorescence reaction. The expression dynamics of endothelium Annexin-II (A2),VE-cadherin,and various tight-junction proteins is detected.The inherent laws are uncovered,concerning the vascular junction complex injury and permeability transition of duck BBB.The interaction of A2,VE-cadherin,and various tight-junction proteins is further studied by using immunoprecipitation,laser confocal microscopy,and immune colloidal gold techniques in vivo and in vitro.The Molecular mechanism on down-regulation of A2 to VE-cadherin,and its decrease to the reassemble of tight-junction proteins is closely investigated. At the same time,A2 regulation to BBB junction complex is verified in vivo by recombination A2 injection through duck pterygoid vein.Together with the last discovery,our results will systematically elucidate the pathological mechanism of DTMUV disease,and theoretically support the prevention and virous vaccine development of the disease.