同位素标记定量代谢组学方法研究及在慢乙肝患者血清肝纤维化标志物筛选中的应用

Patients with chronic hepatitis B virus (HBV) infection with persistently normal or mildly elevated ALT, frequently have significant liver fibrosis or cirrhosis. Currently, liver biopsy needs be performed in these patients to determine the stage of liver fibrosis and treatment regimens. However, this invasive surgery is of high risk and not acceptable for many patients. Therefore, there is a great need to discover new biochemical biomarkers in order to diagnose liver fibrosis at early stage, determine the extent of fibrosis. In this study, we plan to develop workflow to apply chemical isotope labeling liquid chromatography mass spectrometry (CIL LC-MS) for biological studies and discovery a panel of serum metabolites as potential specific and sensitive biomarkers of liver fibrosis. We will perform large-scale metabolomic profiling work of all the serum samples collected from patients with chronic hepatitis B using of 12C-/13C-dansyl chloride (DnsCl) labeling for profiling the amine/phenol submetabolome, 12C-/13C-dimethylaminophenacyl (DmPA) bromide labeling for profiling the carboxylic acid submetabolome and 12C-/13C-dansyl hydrazine labeling for profiling the ketone/aldehyde submetabolome including many sugars. These metabolic information may be used not only for develop a noninvasive model for prediction of liver fibrosis, but also for investigating the possible mechanism on the process of disease development and providing the theoretical and experimental basis on the characteristics of histological abnormalities in a large population of patients with chronic HBV infection.

ALT持续正常或轻度异常的慢乙肝患者存在较高比例显著肝组织损害,肝活检仍是衡量炎症活动度及判定抗病毒指征的重要依据，但因其创伤性难以被多数患者接受。因此，临床上迫切需要无创性指标评估肝组织纤维化程度。本项目拟以慢乙肝肝纤维化预警及早期治疗为突破口，建立基于同位素标记定量代谢组学方法进行生物学研究的标准化流程，并将其应用于慢乙肝患者血清肝纤维化标志物筛选中，获得含“化学标签”（12C/13C-DnsCl、12C/13C-DMPA、12C/13C-DnsHz）的慢乙肝患者血清代谢组，分析其在慢乙肝肝纤维化发生发展中的作用及机制，构建高效的与慢性乙肝患者肝脏炎症分级相关的预测模型并验证，明确其在临床诊断中的作用及价值,为改善该类疾病的诊疗效果提供理论和实验基础。

ALT持续正常或轻度异常的慢乙肝患者存在较高比例显著肝组织损害,肝活检仍是衡量炎症活动度及判定抗病毒指征的重要依据，但因其创伤性难以被多数患者接受。因此，临床上迫切需要无创指标评估肝组织纤维化程度。本项目以慢性乙肝肝纤维化预警及早期治疗为突破口，建立基于同位素标记定量代谢组学技术进行生物医学领域研究的标准化流程。课题首先研究分析前变异因素血清冻融循环、血清存储时间对血清代谢谱的影响,总结一套适用于临床研究的定量代谢组学标准化工作流程；其次,将此技术平台用于慢乙肝患者血清肝纤维化标志物的筛选中，获得含同位素“化学标签”的慢乙肝患者血清定量代谢谱，分析其在慢乙肝患者肝纤维化发生发展中的作用及机制，构建肝脏炎症分级预测模型并验证。基于生信分析表明,慢乙肝患者体内赖氨酸降解通路受到显著影响，其在肝细胞内降解过程中累积产生一系列具有线粒体毒性的代谢物，推测其破坏肝细胞氧化还原稳态，增加肝细胞的氧化压力，激活肝星状细胞转型成纤维细胞，导致胶原蛋白的沉积和纤维组织的生成，继而导致肝纤维化和肝硬化。