呼吸道合胞病毒（RSV）为副粘病毒科肺炎病毒属的单负链RNA病毒，已被认为与儿童急性脑病和CNS症状有关。活化的中枢神经元和小胶质细胞TLRs在多种形式的CNS损害中起重要作用，但RSV如何感染中枢神经元及其损伤的机制，目前尚不清楚。TLR4和新发现的核仁素能识别RSV F蛋白，TLR7和3能识别病毒RNA；中枢神经元和小胶质细胞表面同时表达有TLR4和核仁素。因此本项目拟在细胞模型上运用TLR4/核仁素siRNA干扰和Transwell共培养技术，用激光共聚焦、流式细胞术、分子生物学等方法，检测F-受体的结合和表达、细胞活力和凋亡、炎性因子及病毒增殖水平等，研究F蛋白与其共受体的相互作用、TLR7/3活化、RSV激活小胶质细胞分泌炎性因子对中枢神经元的影响，以阐明RSV感染中枢神经元及其损伤机制。对完善RSV与细胞的相互作用理论，作为分子靶标用于RSV脑病的快速诊断和治疗，具有重要意义。

Respiratory syncytial virus (RSV) is a single negative-strand RNA virus of Paramyxoviridae pneumonia family, has been known to be associated with acute encephalopathy in children, and is related with central nervous system symptoms. Some researches have demonstrated that activation of Toll-like receptors (TLRs) expressed on the central neurons and microglia cells play important roles in various kinds of damages in central nervous system. But the mechanisms how RSV infects central neurons and injury central neuronal cells have not yet been clearly. Recently, we knew that TLR4 and nucleolin, a newly discovered receptor as a functional receptor for RSV F protein, all expressed on the surface of cells could recognize the RSV F protein, respectively. And TLR7 and TLR3 could recognize viral single-stranded RNA and double-stranded RNA produced in the course of RSV replication, respectively. Meanwhile central neurons and microglia cells all express TLR4 and nucleolin receptors. So in the current project we will intend to use siRNA interference for TLR4 and nucleolin receptors and Transwell co-culture technology in the cell models, combining with other methods such as laser confocal microscopy, flow cytometry and molecular biology techniques etc, to detect F-TLR4 and F-nucleolin combination, receptors expression, the vitality and apoptosis of central neurons, the levels of inflammatory cytokines and viral titers. To study the interaction between F protein and its co-receptors, activation of TLR7 and TLR3, and neuronal damage caused by a number of inflammatory cytokines released from RSV activating microglia. We will hope to illustrate how RSV infects central neurons and its injury mechanisms. It will be of great importance in the completing theory of interaction between RSV and host cells and in the rapid diagnosis and effective treatment of human RSV associated encephalopathy by utilizing these molecular targets.

呼吸道合胞病毒（RSV）已被认为与儿童急性脑病和CNS症状有关。活化的中枢神经元和小胶质细胞TLRs在多种形式的CNS损害中起重要作用，但RSV如何感染中枢神经元及其损伤的机制，目前尚不清楚。本项目在细胞模型上运用TLR4/核仁素siRNA干扰和Transwell共培养技术，用激光共聚焦、流式细胞术、分子生物学、病毒学、ELISA等方法，研究TLR4/核仁素活化、RSV F蛋白与受体TLR4/核仁素的相互作用、TLR7/3活化、炎性因子及病毒增殖水平、以及RSV激活小胶质细胞分泌炎性因子对神经元的影响。结果显示，TLR4和核仁素（C23）在神经元表面与F蛋白存在共定位；在病毒感染1~4 h入胞过程中，F蛋白与TLR4和C23结合逐渐增加，而TLR4可结合更多的F蛋白。通过RSV感染和siRNA干扰发现，在mRNA转录和蛋白水平，TLR4和C23在病毒入胞过程中的表达也逐渐升高，4 h达到最高峰；siRNA干扰后TLR4和C23表达虽有上调，但均比RSV感染组同时间点明显下降，且具有统计学差异。这些结果说明，TLR4和C23在识别RSV入胞中具有重要作用。同时发现，RSV感染时TLR3/TLR7/NF-κB蛋白表达升高且具有时间依赖性，阻断TLR4或C23表达可降低TLR3/TLR7/NF-κB的活化过程，而阻断TLR4后的降低更为明显；阻断TLR4或C23表达亦可降低RSV感染N2a细胞的凋亡产生，降低细胞上清中病毒滴度，而TLR4 siRNA组较RSV感染和C23 siRNA组的病毒滴度下降更加明显，表明TLR4在识别RSV中可能具有更重要的作用。ELISA结果显示，IL-6、IL-8和TNF-α炎症因子水平升高具时间依赖性，沉默TLR4或C23表达可减轻RSV感染过程中的炎症反应。Transwell共培养发现，病毒本身和小胶质细胞激活分泌的细胞因子均可导致神经元损伤和凋亡。综上，神经元胞膜表面TLR4和核仁素受体可结合RSV F蛋白，从而识别病毒进入细胞，活化胞内TLR4、C23、TLR3、TLR7、RIG-I、NF-κB表达，使下游炎性因子IL-6、IL-8和TNF-α分泌升高，介导天然免疫反应的产生，导致神经元损伤与凋亡。初步阐明了RSV感染中枢神经元及其损伤机制，对完善RSV与细胞的相互作用理论，作为分子靶标用于RSV脑病的快速诊断和治疗，具有重要意义。