肠道病毒71型感染人脑微血管内皮细胞膜受体的筛选与鉴定

In the late of 1990s, the cases of hand, foot and mouth disease (HFMD) with severe disease of the central nervous system (CNS) which caused by enterovirus71 (EV71) are increased gradually. But, currently, no vaccines or specific antiviral agents are available for EV71.The major reason is the mechanisms of EV71-CNS pathogenesis remain unclear. Our study found: EV71 could infect and replicate in HBMEC (important member of blood brain barrier). Therefore, we speculated HBMEC own EV71 relevant membrane receptors. EV71 strains utilize the receptor to enter host cells and lead to the broken the BBB and infect the CNS. In this study, we adopt classic receptor research methods to explore the membrane receptors in HBMEC on EV71. Furthermore, the screening receptors were identified by "blocking virus infected cells" and "receptor reconstruction" experimental methods. The study will provide a new idea and important theoretical basis for the research of specific receptors blockers or prevention and control of EV71-CNS infection.

20世纪90年代后期，由肠道病毒71型（EV71）感染所引起的手足口病（HFMD）并重症中枢神经系统疾病(CNS)而导致死亡的病例逐渐增多，目前仍无特效抗病毒药物和商品化疫苗问世，主要与EV71致CNS感染的致病机制尚不明确有关。本课题率先研究证实：EV71可以感染血脑屏障主要构成人脑微血管内皮细胞(HBMEC)并能在其中复制。结合病毒感染复制机制我们作出大胆推测：HBMEC存在EV71相关膜受体，EV71可与其结合进而内化并在其中复制，导致了BBB结构破坏从而感染CNS。因此，本项目拟采用经典病毒受体研究方法探明HBMEC内的EV71相关膜受体，并进一步采用"病毒感染细胞阻断"与"受体重建"实验对筛选的受体进行鉴定。本项目将为研究受体特异性阻断剂、防治EV71中枢神经系统感染提供新思路与重要理论依据。

20世纪90年代后期，由肠道病毒71型（enterovirus71，EV71）感染所引起的手足口病并重症中枢神经系统疾病(central nervous system，CNS)而导致死亡的病例逐渐增多，目前仍无特效抗病毒药物问世，主要与EV71致CNS感染的致病机制尚不明确有关。本课题率先研究证实：EV71可以感染血脑屏障主要构成人脑微血管内皮细胞(human brain microvascul cells, HBMEC)并能在其中复制。结合病毒感染复制机制我们推测：HBMEC存在EV71相关膜受体，EV71可与其结合进而内化并在其中复制，导致了BBB结构破坏从而感染CNS。.因此，本项目组①采用经典病毒受体研究手段初步筛选与EV71相关的HBMEC膜受体；②采用流式细胞术、细胞免疫荧光技术验证初步筛选出的膜受体是否在HBMEC膜上表达；③采用RNAi技术、腺病毒包装过表达、RT-qPCR技术手段对筛选出EV71 膜受体进行功能验证。.我们证实：①GST pull-down及LC-ESI-MS/MS初步筛选了人脑微血管内皮细胞上103个与EV71VP1相关的候选膜受体；②采用免疫共沉淀与免疫印迹技术进一步证实GRP78、GSTP1二个候选膜受体与VP1蛋白存在相互作用；③ GSTP1在HBMEC膜上有表达；④GRP78与GSTP1这两个膜受体能调控EV71穿体外BBB过程。.总之，本项目为研究受体特异性阻断剂、防治EV71中枢神经系统感染提供新思路与重要理论依据。