构建HFMD-EV71神经损害MR特异性分子探针的实验研究

Hand, foot and mouth disease (HFMD) is a common infectious disease in children and is charactristed as highly representing philic-nerves, progressing fast after infection, causing severe neurological complications and high morbidity and mortality,enterovirus 71 (EV71) is one of the main pathogen. Hainan is one high-risk areas of EV71 infection. Our preliminary research results showed that MRI can display the lesions site of EV71-HFMD infection and the scope of the damages to the central nervous part, judge prognosis to some extent.But its pathogenesis is not clear, disease progression have often progressed fiercly when conventional MRI detected lesions. This project intends to build specific GoldMag - EV71-VP1-C4 molecular probe of MR in vitro and in vivo experiments of people cynomolgus monkey EV71 infection animal model, combined with the molecular biology technology, tracer EV71 virus proliferation in animal body, histopathological changes and the expression of virus antigen in different organizations, to clarify its selective intrusion, targeted ways to spread, the traceability and descriptive research to further expand as the institutional research.It is expected to seek early accurate diagnosis and reliable inspection methods and specific indicators , for timely and maximizely interventing infection EV71-HFMD to reduce nerve damage and to provide new ideas , which has important theoretical significance and clinical value.

手足口病(HFMD）是儿童常见传染病，肠道病毒71型（EV71）是主要致病病原体之一，具有高度嗜神经特性，感染后病情进展快，易发生严重神经系统并发症，致残率和死亡率高，海南是EV71感染的高发地区。本课题组前期研究结果表明：MRI可显示EV71感染HFMD中枢损害病变的部位和范围，并在一定程度上判断预后，但其发病机制不清，常规MRI检出病变时病程常已进展凶猛。本项目拟构建特异性MR分子探针GoldMag-EV71-VP1-C4抗体探针，通过体外、体内实验，建立人EV71感染食蟹猴动物模型，结合分子生物学技术，阐明EV71选择性嗜神经侵入、靶向性扩散的途径和机制，探讨应用特异性MR分子探针早期诊断EV71感染中枢损害的可能性，以期为临床早期干预治疗，最大程度减轻患儿神经损害提供新方法，具有重要理论意义和临床价值。

拟构建特异性MR分子探针GoldMag-EV71-VP1，通过体外、体内实验，建立人EV71感染小鼠动物模型，结合分子生物学技术，纵向示踪EV71在动物体内病毒增殖、组织病理变化以及病毒抗原在不同组织的表达，以阐明其选择性嗜神经侵入、靶向性扩散的途径。本项目从手足口病患儿咽拭子中分离出流行的EV71毒株，原核表达纯化该毒株EV71 VP1衣壳蛋白，以此蛋白为免疫原制备VP1的特异性小鼠单克隆抗体。Fe磁性颗粒标记纯化的单克隆抗体11R12-IgG，磁化标记的单抗11R12-IgG运用与protein-A/G-beads共孵育及SDS-PAGE分析测量抗体吸附效率。然后用ELISA测试11R12-IgG-beads与纯化的VP1抗原的结合能力。最后MRI信号分析磁性颗粒标记的抗体在细胞水平上检测病毒感染分布的能力。病毒毒株纯化扩大培养后进行小鼠攻毒实验，筛选到一株病毒EV71 C4-WH在10^7pfu/ml时能使小鼠出现典型的神经症状。用此病毒株在12-14日龄乳鼠上连续适应5代，成功建立了2周龄BALB/c小鼠的EV71模型，滴定小鼠肝脏，脾脏，大脑及肌肉组织的EV71病毒滴度，观察不同攻毒日龄的小鼠的病毒在组织中的分布。项目资助发表期刊论文4篇（其中SCI论文2篇，中文核心期刊论文1篇，中文统计源期刊论文1篇）及综述1篇。项目投入经费47万元，支出23.05万元，各项支出基本与预算相符。剩余经费23.95万元，剩余经费计划用于本项目研究后续支出。