25-羟基胆固醇在登革热发病及早期诊断中的作用研究

The consequences of DENV infection are very complex. Currently, some warning signs have been found for predicting severe dengue disease. CH25H is an antiviral protein induced by type I IFN, and can catalyze 25HC production in the intrinsic pathogen response. Recently, we found that CH25H gene was up-regulation in the host cells infected by dengue virus or Zika virus, and 25HC had the antiviral efficacy against dengue virus in vitro. However, It is unclear whether 25HC exists or not in the serum of dengue patients, and we do not understand the function of 25HC for the dengue diagnosis. In this project, the cohort of patients classified according to the severity of dengue disease will be recruited in collaboration with Guangzhou 8th People’s Hospital, and blood samples from each patient will be daily collected during the period of hospitalization. Then, the metabolite 25HC will be detected in all serum samples through the metabolomics and analytic software, and the kinetic of this marker will be carefully investigated. The correlation between 25HC and the severity of disease will be determined using t test analysis. Furthermore, the antiviral mechanism of 25HC during the process of infection by dengue virus will be determined through the internalization assay. Finally, it will be decided whether the inflammatory response enhances or not during the process of infection by dengue virus through the flow cytometry and CH25H deficient mice model. Our project will provide the data for making 25HC as an early diagnostic marker of severe dengue, and it is of highly importance for diagnosis and therapy for dengue. Our project will also clarify the function of 25HC during the infection by dengue virus, and help us understand the pathogenesis of dengue.

登革病毒感染导致的临床表现非常复杂，近年来，一些决定登革热疾病转归的风险因子相继被发现。胆固醇-25-羟化酶（CH25H）是一种由I型干扰素介导产生的抗病毒蛋白，可催化胆固醇转变成25-羟基胆固醇（25HC）。最近我们发现，登革病毒和寨卡病毒感染可显著上调宿主CH25H基因的表达，而且25HC在细胞水平上具有显著的抗登革病毒效果。但是，25HC在登革热患者血清中是否存在？其对登革热的早期诊断是否具有意义？目前还不清楚。本研究将建立登革热和重症登革热患者队列，连续采集患者发热后不同时间点的血清标本，利用代谢组学方法，明确血清中25HC的动力学特征，及其与登革热严重程度的相关性。最后，采用细胞内摄实验，流式细胞技术等方法，及CH25H基因缺陷小鼠确定25HC在登革病毒感染过程中调控宿主反应的分子机制。该项目的开展将为登革热的早期诊断提供新的生物标志物，对于阐明登革热的发病机制亦具有重要意义。

登革热是一种蚊子传播的病毒性疾病，近年来迅速蔓延到世界卫生组织下设所有地区。我国是登革热的重要流行区，历史上广东、福建、湖北、海南都曾暴发过规模超过百万人的登革热流行。2014年登革热在我国广东再次暴发流行，引起此次大流行的登革病毒株的病原学研究表明登革热在我国已经形成了地方性流行，严重威胁我国的公共卫生安全。在发病早期对登革热患者进行准确的病员分类，对于有效利用医疗资源，减少由于延误治疗造成的病情恶化具有重要意义。本项目的研究内容包括建立登革热患者和重症登革热患者队列，检测患者血清中25-HC等代谢物的含量，明确其动力学特征，及其与登革热严重程度的相关性，明确25-HC等体内代谢分子拮抗登革病毒感染的作用机制。本项目首先收集了重症登革热患者14例、非重症患者22例的入院血清样本，同时按照重症患者年龄及性别匹配健康对照组26例。结果发现犬尿酸、肉碱 C12:3、5'-脱氧腺苷和D-果糖-6-磷酸二钠盐可作为可能的预警重症登革热的生物标志物。随后，我们收集了登革热患者动态血清样本148份，即轻症患者72份和重症患者48份，同时纳入28例相配伍的健康体检者血清。采用液相色谱-质谱联用系统检测血清样品中19种氧化胆固醇代谢物的含量。结果发现，登革病毒感染后，可导致机体的氧化胆固醇代谢物FF-MAS、T-MAS、24S-25-EPOXY-CHO、7b-OH-CHO、22S-OH-CHO、6K-5aOH-CHO和7a-OH-cholestenone的浓度发生显著改变，其中，恢复期7a-OH-cholestenone和24S-25-EPOXY-CHO的显著减少与重症登革密切相关。进一步对25-HC等代谢分子的抗病毒作用机制进行了初步探索，发现25-HC和7a-OH-cholestenone可有效抑制登革病毒的复制。以上研究为登革热的早期诊断提供了新的生物标志物，对于指导登革热的临床诊疗具有重要意义，亦为揭示登革热的发病机制奠定了基础。