基于动态生物网络的慢性乙型肝炎典型证候病证关系分子机制研究

Traditional Chinese medicine (TCM) treatment is regarded as a safe and effective method for Chronic Hepatitis B (CHB), which requires a traditional diagnose method to differentiate the TCM syndrome. However, the Disease-Syndrome Mechanism of CHB still is inexplicit. Molecular biological network was used for many chronic disease studies, which exhibits a new route for CHB research. In previous studies, we found that the miRNA expression levels are great differences among the TCM syndromes of CHB. The miRNA-target dynamical network suggested that the prognosis of Excessive-deficient syndrome and Deficient syndrome might poorer than Excessive syndrome in CHB patients. The expression profile of serum protein also implicated that the CHB syndromes have distinct separation tendency. Based on these findings, we hypothesized the relationship between Disease and Syndrome of CHB is complex, the different "Syndrome" might causes different "Disease" in prognosis, and the related molecular mechanism of Disease-Syndrome also different. In the present project, the representative and common clinical Excessive syndrome (Liver-gallbladder dampness heat syndrome, LGDHS),Liver depression and spleen deficiency syndrome (LDSDS), and Deficient syndrome (Liver-Kidney Yin Deficiency Syndrome, LKYDS) of CHB patients were studied, the Occult syndrome (Differentiation of Syndrome Unavailable, DSU) and healthy donors act as control. We first deteced the miRNA, mRNA, and protein expression profiles from CHB samples. Then, the dynamical molecular networks of CHB syndromes were constructed using TCM special biological algorithm. Furthermore, the potential molecular biomarkers of CHB syndromes were screened by bioinformatics, and explore the correlation between the biomarkers and CHB syndromes based on qPCR/Western Blot validation. The project would help uncover the molecular mechanism of Disease and Syndrome of CHB, and provide clues to TCM treatment of CHB patients.

中医辨证治疗慢性乙型肝炎(CHB)有独特作用，但CHB病证分子机制尚未阐明。近年来生物网络技术为CHB研究提供了新方法。我们前期研究发现：CHB不同证候miRNA表达差异明显，分子网络显示虚证和虚实夹杂较实证预后更差；蛋白表达谱显示CHB证候间有明显分离趋势。据此我们推测：不同"证"对应的CHB"病"可能产生不同预后，其对应的病证机制也不同。本课题拟在此基础上，以临床常见且具有代表的CHB典型实证（肝胆湿热证）、虚实夹杂（肝郁脾虚证）、虚证（肝肾阴虚证）为切入点，以疾病中隐证型（无证可辨）和健康者为对照，检测其miRNA/mRNA/protein表达谱信息，设计中医药特色算法，构建CHB典型证候相关动态分子网络，采用生物信息学方法筛选潜在分子标志物，并运用qPCR/Western Blot 验证与CHB典型证候的关系。本课题将有助于揭示CHB病证分子机制，为慢性乙型肝炎中医治疗提供新线索。

慢性乙型肝炎（CHB）是由乙型肝炎病毒引起的一种常见世界性疾病，我国是乙肝病毒感染大国，慢乙肝已成为严重影响我国居民健康的一大临床与公共卫生问题。尽管中医辨证治疗慢乙肝有着独特作用，但慢乙肝不同证候间的动态变化及病证关系机制尚未完全阐明。本项目通过对CHB三种典型证候转录组（miRNA、mRNA）及蛋白组（protein）数据进行检测及分析，分别获得了CHB肝胆湿热证（实证）、肝郁脾虚证（虚实夹杂证）、肝肾阴虚证（虚证）miRNA、mRNA及protein的生物物质基础。利用构建的动态生物网络筛选了CHB三种典型证候miRNA、mRNA及protein的特征生物物质；基于RT-PCR及western blot方法对特征生物物质进行分子生物学验证，获得了CHB肝胆湿热证（实证）、肝郁脾虚证（虚实夹杂证）、肝肾阴虚证（虚证）的潜在分子标志物，包括9个miRNA，4个mRNA及4个protein。这些潜在标志物在CHB典型证候分类及证候由实转虚演化过程具有重要作用。进一步对CHB三种典型证候的动态生物网络结构进行分析，结合四诊信息、理化信息、生物学功能及分子生物学验证等信息，发现虚实夹杂证是CHB证候由实转虚演化过程的一个重要临界点，在这一阶段，大量生物事件集中爆发，如细胞周期、细胞凋亡与增殖、肿瘤发生、肿瘤侵袭、迁徙和转移等；同时还包括p53信号通路和TGF-β信号通路等许多调控事件发生。这些生物事件在CHB疾病向“右”或向“左”的进展路线中可能发挥重要作用。通过对这些生物事件的有效调节和/或控制， CHB证候特征有可能朝着“正确”的方向演化。至少，它可以帮助延缓CHB疾病向恶性方向发展的演变时间，并且对于预后有一定的改善。本研究从miRNA、mRNA及protein三个层面探索了CHB典型证候潜在分子标志物及其可能病证关系，具有一定的临床应用预期及较好的学术应用价值。