肠道病毒71型诱导树突状细胞自噬引起免疫功能调变的研究

In recent years, foot and mouth disease increasing epidemic in china. Enterovirus 71 (EV71) is the causative agent of human diseases with distinct severity, from mild hand, foot andmouth disease to severe neurological syndromes, such as encephalitis and meningitis. The lack of understanding of the mechanism of immune response induced by EV71 infection as well as lack of efficient vaccine and drugs against this virus impedes the control of EV71 infection. Recently, more researches have been showed that cellular immune response in China has been experiencing an increasing epidemic of hand foot and mouth disease(HFMD) in the past years with a high incidence rate. Enterovirus 71 (EV71) is the major causative agent for HFMD. Up to now, the mechanism of EV71 induced immune response is still uncertain and there is no preventive vaccine and specific anti-EV71 drugs available. Studies have shown that T-cell mediated immune response in EV71 infected children was reduced and the reduction state is correlated with the disease severity of the patients. Dendritic cells (DCs) play important roles in triggering T cell responses as professional antigen presenting cells. We speculate that the abnormal T cell responses might be resulted from the functional changes of DC induced by EV71 infection. This project aims to study the immune function modulation of DC in EV71 induced T cell responses, revealing the key pathogenic mechanism of EV71 infection induced host immune responses, paving the way to identify potential candidate targets for developing anti-EV71 infection drugs or supplying new information to develop new strategies for HFMD control.

近年来，手足口病在我国的流行呈快速上升趋势，其发病率高居不下。肠道病毒71型（EV71）是引起手足口病的主要病原体，但EV71感染所引发的免疫应答机制尚不明确，到目前为止尚无有效的疫苗及特效抗病毒药物用于手足口病的预防和控制EV71。以往研究显示EV71感染患儿病情的严重程度与机体细胞免疫功能的降低程度高度相关。树突状细胞（DC）作为机体专业的抗原提呈细胞将抗原信息提呈给T细胞以启动细胞免疫反应。我们推测EV71感染引起的T细胞功能异常可能与DC细胞的功能改变有关。本项目拟通过研究EV71诱导DC细胞发生自噬后导致DC细胞的功能调变，初步阐明DC细胞在抗EV71感染免疫中所发挥的作用，进一步明确EV71的致病机制及机体对EV71的抗病毒免疫或EV71的免疫逃逸机制，为寻找新的抗EV71感染的的药物靶标或制订防治措施提供参考信息。

近年来，随着对自噬的深入研究，发现自噬可以作为机体天然免疫的一部分参与到抗感染免疫中，但病原体也可以通过调控自噬来完成其感染和致病过程。本项目通过研究发现EV71感染可诱导DC细胞发生自噬、凋亡及导致DC细胞分泌炎性细胞因子的水平增加，说明EV71感染可通过DC细胞引起炎性反应，为其临床EV71感染所致脑干脑炎等炎性疾病提供细胞水平实验研究的支持。同时，我们观察了DC2.4、RD-A、GES-1、Hela等细胞对EV71感染的敏感程度，选择RD-A细胞为模型进行EV71感染细胞的转录组测序。从测序结果我们观察到在感染后小于 90分钟的时候细胞内基因表达水平发生明显改变，180分钟变化达到峰值，细胞内相应信号通路改变异常。但360分钟时基因表达差值反而开始下降，western-blot结果显示360分钟时细胞内已经合成病毒的衣壳蛋白VP-1， LC-3出现明显型别转换，说明此时病毒通过促进细胞自噬，并逃脱了细胞防御系统对它的制约，并已经开始在细胞内大量复制。我们正在对测序结果进行深度挖掘，为下一步深入了解病毒对感染细胞的致病机制及细胞的抗病毒调控提供了参考。接下来，我们通过生物信息学手段和分子生物学方法找到了EV71感染宿主细胞后发生变化的相关联的microRNA，并选择两种自噬蛋白作为其作用靶点进行检测研究。结果显示这一microRNA的表达与两种自噬蛋白的mRNA表达水平呈负相关，为进一步阐明EV71诱导自噬的机制提供参考。另外，我们在研究中发现病毒受体PSGL-1可参与EV71的感染，并且参与诱导了细胞自噬，为明确EV71诱导自噬的机制提供参考。总之，我们从细胞水平、蛋白水平和基因水平等多方面观察EV71对细胞自噬及免疫功能调变的影响，并得到了初步的结果，发现了EV71感染过程中参与诱导细胞自噬的蛋白、microRNA，观察到了EV71感染引起细胞自噬、凋亡及免疫调控的改变，得到了EV71感染引起敏感细胞转录组改变的数据，为今后进一步深入研究EV71感染的致病机制提供了基础。今后，我们将在本课题已有数据基础上开展进一步深入的研究工作。