多发性硬化湿热证重组cDNA表达克隆标记物的识别及其病证关联特征的研究

In multiple sclerosis, about 50% of recurrent-remission (RRMS) patients are converted to secondary-progressive (SPMS) within 10 years. Traditional Chinese medicine (TCM) considered that damp-heat characterized by turbidity heavy and viscosity is an important factor in MS recurrent. However, the objective basis or mechanisms for evolution of traditional Chinese medicine (TCM) syndromes was insufficient. Serum biomarkers are objective indicators of modern medicine assisted diagnosis and prediction of disease progression in MS. Accordingly, if the objective biological basis of MS damp-heat syndrome could be revealed from the level of serum biomarkers, the objectification for TCM treatment will be remarkably promoted. It is noteworthy that recombinant cDNA expression clone serology identification technology (SEREX) technology has been shown to be an effective method for screening new serum heterosexual biomarkers in MS. Therefore, SEREX technology was employed to screen damp-heat syndrome and Non- damp-heat syndrome related serum biomarkers in RRMS and SPMS, which will be more helpful to obtain related serum biomarkers and provide objective indicators for MS damp-heat syndrome and syndromes evolution. This project will provide a new perspective and method for the study of MS damp-heat syndrome theory, and lay favorable foundation for the precise treatment and exploration of new targets of TCM for patients with MS damp-heat syndrome.

多发性硬化(MS)的复发缓解型(RRMS)患者中约50%在10年内转变成继发进展型(SPMS)，中医认为MS湿热证患者常反复发作、迁延不愈，具有湿性重浊粘滞的特征。而中药在MS防复发中作用明显，但核心证候研究不足、缺乏客观指标，无法突显中医优势。血清生物标记物是西医辅助诊断MS、预测疾病进展的客观指标，若能从血清生物标记物层面揭示MS湿热证患者的生物学基础，亦可阐明MS湿热证候特征及“病证”演变趋势，为中医在治疗、防复发等方面提供佐证。重组cDNA表达克隆血清学鉴定技术(SEREX)是筛选MS特异性生物标记物的有效方法，因此本研究采集RRMS和SPMS的湿热证、非湿热证患者四诊信息和血清样本，应用SEREX技术筛选RRMS和SPMS的湿热证、非湿热证相关靶抗原，更有助于获得MS证候相关客观指标。本项目为研究MS核心证候提供新的思路和方法，为中医精准治疗、探索并寻找中药新靶点等方面提供依据。

多发性硬化（MS）是一种典型的中枢神经系统炎性脱髓鞘性自身免疫性疾病，该病临床类型复杂，复发缓解型多发性硬化（RRMS）最常见，占MS疾病类型的80%以上，病程呈复发-缓解、阶梯样进展，具有高复发率和致残率的特点。中医药研究则通过临床实践，发现湿热之邪是MS反复发作、迁延不愈的重要因素，目前仍然缺乏MS湿热证及证候演变的客观性依据。血清生物标志物是现代医学辅助诊断MS、预测疾病进展的客观指标。若能从血清生物标志物层面揭示MS湿热证患者的生物学基础，亦可阐明MS湿热证候特征，为中医在治疗、防复发等方面提供佐证。本研究首先利用重组表达cDNA克隆的血清学分析技术（SEREX）以及蛋白质组学技术共筛选出候选标志物60余个，进一步利用单分子阵列分析技术检测受试者潜在候选生物标志物：血清神经丝轻链蛋白（NfL）、可溶型程序性死亡受体1 （sPD-1）、可溶型程序性死亡配体1 （sPD-L1） 水平，并采用Spearman法对RRMS患者血清NfL、sPD-1、sPD-L1水平之间的相关性及其与病程、发作次数、EDSS评分的相关性进行分析。结果显示，RRMS组患者血清NfL、sPD-1、sPD-L1水平显著高于健康组（P＜0.05或P＜0.01）；肾虚湿热证亚组患者血清NfL、sPD-1、sPD-L1水平均高于肾虚非湿热证亚组患者（P＜0.05或P＜0.01）。RRMS组患者血清NfL水平与sPD-1、sPD-L1水平均呈正相关（P＜0.01），sPD-1水平与sPD-L1水平呈正相关（P＜0.001）；RRMS组患者血清sPD-1水平与发作次数呈正相关（P = 0.030）。该研究表明，RRMS缓解期患者血清NfL、sPD-1、sPD-L1显著升高，且肾虚湿热证患者较肾虚非湿热证患者升高程度更为明显，提示RRMS肾虚湿热证患者可能存在更为严重的免疫炎症和神经损伤。本项目为MS湿热证的核心病机研究提供新的思路和方法，为中医精准治疗、明确中药新靶点等方面提供依据。

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