基于卫气营血辨证的EBV致发热患者中医证候及血浆microRNA表达相关性研究

Based on EB virus infection is associated with warm disease in TCM, This subject aims at studying on TCM syndrome characteristics of the fever patients caused by EBV, screening the biological indicators for the diagnosis of syndrome, researching the pathogenic mechanism. Collect the medical record data of outpatient and hospitalized patients in fever infected by EBV from January 2020to June 2021, according to western medicine diagnostic criteria determine the disease；on the basis of warm disease wei qi ying blood syndrome differentiation standard and warm disease theory, determine the diseases' syndrome types; according to the liver and kidney function, immune function, indexes of peripheral blood lymphocyte differentiation antigen observe symdrome's difference in molecular biology level. Combined with the warm disease’s onset theory analyse the relevance between time, district, occupation factors and the symdrome. Through high-throughput HiSEQ technology choose early the miRNA with significant differences on different syndromes. Using PCR technology to validate its sensitivity and specificity as indicators of syndromes. Drawing ROC curve, define the critical value distinguishing syndrome. To investigate how the expression of miRNA regulate the inflammatory response and cellular immunity in EBV infection in vitro, and explore the scientific nature of miRNA.This study can provide reference for TCM diagnosis and the establishment of relevant therapeutic targets for the further intervention on fever infected by EBV.

基于EBV感染致发热与中医温病相关性，探析EBV感染致发热患者中医证候特征，筛选证候诊断的生物学指标，进一步研究其致病机制。采集2020年1月至2021年6月确诊为EBV感染致发热患者病例90例，依据西医诊断标准明确疾病种类，依据温病学卫气营血理论与辨证标准确定患者卫气营血阶段及具体证候类型；根据患者microRNA、肝肾功能、免疫功能、外周血淋巴细胞分化抗原等指标，结合温病学发病理论分析患者的症状、体征、实验室检测指标与中医证候的相关性；通过高通量HiSEQ技术筛选随证候不同而有显著差异的血浆microRNA，经PCR技术验证已筛选的microRNA作为证候诊断生物学指标的灵敏性与特异性，绘制ROC曲线，界定区分证候的临界值；对已筛选出的microRNA进行过表达或抑制，考察其对EBV诱导的细胞免疫炎症应答影响。本研究将为该类疾病中医证候的基因诊断和确立证候相关治疗靶点提供借鉴。

EB病毒（Epstein-Barr Virus，EBV）是一种感染人类B淋巴细胞的疱疹病毒，可能引发一系列具有发热性质的急性病症或淋巴组织增生性疾病。该类病证属于中医学“温病”范畴，综合运用温病学理论，寻找中医证型与现代临床实验室检测指标的明确关联，筛选证候鉴别的生物学指标，对于中医药干预治疗以提高该类疾病的临床疗效具有重要意义。本项目主要研究内容：1.分析EBV致发热不同证候间实验室指标相关性；2.筛选可能具有EBV致发热患者不同疾病、不同证候间鉴别意义的EBVmicroRNA并验证其可行性；3.通过体外Raji细胞、NCI-BL2009细胞实验，探讨所筛选的EBVmiRNA炎症干预机制。分析70例EBV致发热患者病史资料、实验室生化指标，建立与EBV发热患者中医卫气营血辨证的关联，结果表明：EBV致发热患者中医证候为气分证时，与PCT升高、免疫应答激活、肝功能异常等具有强关联性；营分证和气营两燔与贫血、免疫反应、肝功能异常、脾肿大等具有强关联性；血分证与PCT升高、炎症反应、贫血等具较强关联性。以中医视角分析，该病主要病位在脾，气分证阶段以湿热内蕴困脾为主，兼以气阴两虚；若伴有贫血症状者，与心、脾、肝相关；以肝功能异常及肝脾肿大为主要表现者，病位多在肝、脾、肺，病性为痰湿伴有气虚；以免疫指标改变（CD3+T升高，淋巴细胞亚群比例降低）为主要关联因素者病位多位于淋巴、肝、胆，病邪性质为暑湿和痰热。我们筛选出具有该类疾病病种、证候鉴别诊断价值的三个miRNA，分别为hsa-miR-320d、EBV-miR-BART22、EBV-miR-BART2-3p，在hsa-miR-320初步判断EBV感染的基础上，EBV-miR-BART22可能作为气分证与血分证的鉴别指标，EBV-miR-BART2-3p可能作为气分证、营分证与血分证的鉴别指标。通过体外细胞实验，发现EBV-miR-BART22与EBV-miR-BART2-3p均有一定抑制NF-ΚB p65、IL-10表达的趋势；EBV-miR-BART22具有促进TNF-α、JNK、IL-1β、ERK表达的趋势；EBV-miR-BART2-3p则具有抑制TNF-α、JNK、IL-1β、ERK表达的趋势。此研究为EBV感染致发热中医证候标准化及中医药辨证论治方案的拟定提供参考。