HEV感染致脑组织损伤的机制研究

In this study, using Mongolian gerbils and rabbits as experimental animals, the application of Transwell migration assay and in vitro culture technique for cerebrovascular endothelial cells and glial cells , combined with confocal microscopy techniques, ultrastructural pathology, immunohistochemistry, immuno-electron microscopy, western bloting and PCR techniques, the overall goal of this study is focused on pathogenesis and mechanism of the HEV invading the brain nerve tissue and resulting in injury of brain nerve cells and interstitial tissue。This study will centre on answers to questions " how HEV to break through the blood brain barrier into the brain tissue". The qualitatively and quantitatively protein expressions of ORF2 and ORF3 in brain tissue will be observed in HEV infection process, meanwhile, the damage effect on structure and function of brain nerve cells, cerebrovascular endothelial cells and glial cells of experimental animals infected with HEV will be studied systematically，as well as both qualitative and quantitative detection on signal molecules related to inflammation on the surface of cerebrovascular endothelial cells and glial cells will be carried. The provenge reveals systematically and comprehensively the mechanism of HEV replication, assembly and release in brain nerve cells and the pathogenesis of brain injury caused by hepatitis E virus infection at the cellular and molecular level. This research provides theoretical basis for further study on diagnosis and preventative treatment of HEV, which reveals the pathogenesis of HEV infection from a new perspective.

本研究以长爪沙鼠和家兔为实验动物，应用Transwell 小室迁移试验和脑血管内皮及神经胶质细胞体外培养技术，同时结合激光共聚焦显微镜观察技术，运用超微病理学、免疫组化和免疫电镜技术、Wester Bloting和PCR技术，总体研究目标锁定于HEV侵入脑组织并造成脑神经细胞及间质组织损伤的发病过程及其机制，重点围绕"HEV如何突破血脑屏障进入脑组织"这个核心问题展开观察研究。在观察HEV感染过程中ORF2和ORF3的蛋白表达在脑组织细胞内的定性和定位的同时，系统观察HEV对脑神经细胞、血管内皮和胶质细胞结构和功能的损伤作用，并对脑血管内皮和胶质细胞膜上的炎症相关信号分子进行定性、定量的观测，从一个新的视角在细胞和分子水平上系统全面位地揭示HEV在脑神经细胞内的复制、装配和释放的机制及HEV感染引起脑组织损伤的机制，为深入揭示HEV的发病机理及HEV的防治提供新的科学依据和新的研究思路。

本研究以长爪沙鼠和家兔为实验动物，同时应用Transwell小室迁移试验和脑血管内皮及神经胶质细胞体外培养技术，运用超微病理学、免疫组化和免疫电镜技术、Wester Bloting和Real-time PCR技术，重点围绕“HEV如何突破血脑屏障进入脑组织”这个核心问题展开观察研究。结果如下：.腹腔注射给沙鼠接种HEV后，在急性感染期脑和脊髓中均可检测到HEV正链及其负链RNA；脊髓中的病毒载量高于脑；免疫组织化学观察发现HEV ORF2抗原阳性信号主要分布于血管周围、脉络膜、室管膜上皮及一些神经元和神经胶质细胞胞浆内；组织病理学观察见脑和脊髓出现典型的病毒性脑脊髓炎的变化。超微病理学观察结果显示脑及脊髓细胞胞浆基质减少，细胞器数量减少，线粒体肿胀，嵴断裂；神经髓鞘变性，毛细血管内皮细胞肿胀，结构紊乱不清，内皮细胞之间连接复合物结构缺失。通过腹腔注射途径给兔感染猪源sHEV-4后可在兔脑脊髓中检测到HEV抗原，免疫组织化学和组织病理学观察及荧光定量PCR分析结果均与sHEV-4感染沙鼠的结果基本一致。sHEV-4感染沙鼠试验及sHEV-4接种脑微血管内皮细胞体外试验均证实，HEV感染可导致脑、脊髓微血管内皮细胞多种紧密连接相关蛋白表达量显著减少（P＜0.05或P＜0.01），以致脑、脊髓血管内皮的通透性增加，HEV可以穿过血脑屏障侵入脑脊髓实质中。sHEV-4接种脑微血管内皮细胞Transwell实验结果显示，血管内皮细胞对HEV的内吞及转胞吞作用可以被网格蛋白抑制剂所抑制。western-blot检测结果显示接种HEV的脑微血管内皮细胞中ATP5A1表达量减少而NOX4表达量增加。.上述研究结果显示，sHEV-4可能通过网格蛋白介导的内吞作用进入血管内皮细胞，影响内皮细胞ATP合成引发内皮细胞氧化应激，导致血脑屏障损伤，并通过跨细胞转运以及细胞旁途径穿过血脑屏障结构从而感染脑和脊髓的实质组织，引起小胶质细胞和星形胶质细胞活化增殖，释放促炎因子TNF-α和IL-1β等，这些病变引发了脑和脊髓组织炎症反应，同时激活了细胞线粒体凋亡途径，最终导致脑和脊髓组织的损伤。.本研究为深入揭示HEV的发病机理及HEV的防治提供新的科学依据和新的研究思路。