ADP核糖基化因子6介导EV71感染脑微血管内皮细胞的作用机制研究

The central nervous system infections caused by enterovirus 71(EV71) are the leading causes of death in hand-foot-mouth disease. The mechanisms of breaking through the blood-brain barrier to infect the brain cells are the hotspot in the field of EV71 pathogenesis. Brain microvascular endothelial cells (BMEC) are the first defense against EV71 crossing the blood-brain barrier, however, the mechanisms of virus infection remains unknown. A small interfering RNA library was used in our early study to screen 140 host proteins with membrane trafficking functions. ADP ribosylation factor 6(ARF6) was identified as the key molecular in mediating EV71 infection in BMEC. Our previous work shows that EV71 can specifically utilize ARF6 to invade BMEC and the invasion pathways in BMEC are significantly different from in other tissues and organs. ARF6 is located in cell plasma membrane and endosome membrane, activity of which depends on the conversion of its association with GTP or GDP. This applying project plans to illustrate the molecular and cellular mechanisms of ARF6-mediated EV71 infection in BMEC through exploring the role of ARF6 in transmission of the virus into cells and regulation of the tight junctions between cells, on the bases of established EV71 infection model in vitro and in mouse. This project helps not only identify the key factors and mechanisms of EV71 breaking through the blood-brain barrier, but also provide new targets for the prevention of EV71 infection and the development of anti-EV71 drugs.

肠道病毒71（EV71）引起的中枢神经病变是手足口病患者死亡的主要原因，其突破血脑屏障建立脑细胞感染的机制是领域研究热点。脑微血管内皮细胞（BMEC）是EV71突破血脑屏障的第一道防线，但感染机制仍不清楚。我们前期利用干扰RNA文库，筛选了140个具有膜运输功能的宿主蛋白，发现介导EV71感染BMEC的关键分子-ADP核糖基化因子6（ARF6）。ARF6位于细胞质膜和内体膜，其活性主要依赖于与GTP或GDP结合的转换。前期工作显示：EV71特异性利用ARF6入侵BMEC，且入侵途径显著区别于其他组织细胞。本课题拟在上述基础上，利用已建立的EV71体外模型和小鼠模型，通过探究ARF6在转运病毒入胞和调控细胞间紧密连接结构的作用特点，阐明其参与EV71感染BMEC的分子和细胞机制。课题实施不仅有助于确定介导EV71突破血脑屏障的关键宿主因子及作用机制，还可为EV71感染防治药物研发提供新靶点。

肠道病毒71型（EV71）引起的中枢神经系统（CNS）病变是手足口病患者死亡的主要原因，其突破血脑屏障（BBB）建立中枢神经系统感染的机制一直是本领域的研究热点。脑微血管内皮细胞（BMEC）是EV71突破血脑屏障的第一道防线，但感染机制仍不清楚。.本课题组前期通过干扰RNA文库，对140种与跨膜转运、运输相关的分子进行筛选，发现ADP-核糖基化因子6（ARF6）下调可显著抑制EV71感染，ARF6位于细胞质膜和内体膜，其活性主要依赖于与GTP或GDP结合的转换。为探究ARF6在EV71感染BMEC中的作用机制，我们利用ARF6基因敲除的人脑微血管内皮细胞（HBMEC）株，建立体外EV71感染血脑屏障模型，野生型及基因敲除小鼠EV71感染模型用于鉴定分析ARF6在EV71感染HBMEC、突破BBB中的作用机制。研究结果显示， EV71特异性利用ARF6入侵BMEC，且入侵途径显著区别于其他组织细胞。ARF6通过ERK-Rho GTPase信号通路参与了EV71病毒的感染与入侵过程，其参与了细胞间紧密连接蛋白的分布及表达量调控，可在EV71感染中改变BBB通透性，在CNS感染中起促进作用。动物实验表明， ARF6在EV71感染BMEC和突破BBB中起到关键作用，其抑制剂NAV-2729能在小鼠体内抑制病毒感染建立,为EV71的感染防治提供了新的作用靶点。.该实验项目使我们对EV71感染CNS、入侵BBB的靶点与作用机制有了深入认识，也为研制出新型有效的抗EV71药物提供了新思路。