【作者】 林景超；

【导师】 贾伟； 阎超；

【作者基本信息】 上海交通大学 ， 药理学， 2008， 博士

【摘要】 肝脏是机体最重要的代谢器官之一,而化学性肝损伤是引发多种肝脏疾病的重要始动环节。因此,针对化学性肝损伤的发病机制以及药物干预效应的研究,对慢性肝病的防治具有十分重要的意义。动物实验是研究化学诱导急性肝损伤发病机制的必经之路,在研究肝脏疾病发病机制及抗肝脏疾病药物的过程中,选择有科学性、实用性、重现性的肝损伤动物模型的意义亦十分重要。在肝损伤的实验研究中,四氯化碳(CCl4)诱导的化学性肝损伤和酒精性肝损伤模型是比较常见的两种动物模型。本课题将利用建立的代谢组学方法,分别对两种肝损伤模型动物的代谢特征开展研究,并利用代谢组学方法对药物干预效应进行评价。同时,在动物模型研究的基础上,本课题进一步尝试利用代谢组学方法,对临床原发性肝癌(PLC)患者的代谢特征进行分析,探索将代谢组学方法应用于临床疾病机制研究的新思路。本课题首先建立了基于色谱质谱联用技术的肝组织的代谢组学研究方法。在此基础上,重点考察了CCl4及酒精诱导的急性肝损伤大鼠尿液及肝组织的代谢谱变化,并从代谢水平观察甘利欣对CCl4诱导化学性肝损伤的保护作用及其机制。同时,在整体代谢水平上分析了临床肝癌病人与正常对照人群的代谢谱差异。本课题的主要内容和结果如下:1.建立了基于氯甲酸乙酯衍生化(ECF)的气相色谱质谱联用(GC/MS)分析以及基于三甲基硅烷(TMS)衍生的气相色谱飞行时间质谱(GC/TOFMS)分析的肝组织代谢组学研究方法。针对前者的分析特点,采用高氯酸乙醇两步提取法提取肝组织中的小分子内源性代谢物,后者采用氯仿/甲醇/水的混合溶剂进行两步提取。两种分析方法均经方法学验证表明重复性好、灵敏度高、精密度高,适用于肝组织的快速、高通量检测。2.采用ECF衍生、GC/MS分析的代谢组学研究方法,尝试着从整体代谢水平上考察大鼠在CCl4诱导急性肝损伤过程中尿液及肝组织中内源性小分子代谢物谱的变化,并观察甘利欣预防性给药对CCl4诱导急性肝损伤的保护作用。结合多维统计方法,发现肝损伤大鼠尿液和肝组织样本中小分子内源性代谢物发生了明显变化,并找到使得正常组和模型组区分开的相关差异性代谢物,且尿液和肝组织样本代谢谱所提供的信息相互补充,经初步分析CCl4诱导的急性肝损伤与三羧酸循环、部分氨基酸代谢等高度相关。3.以酒精诱导的大鼠急性肝损伤模型为研究对象,采用ECF衍生、GC/MS分析的代谢组学研究方法检测了大鼠尿液及肝组织中内源性小分子代谢物谱的模式变化情况,结果发现蛋氨酸代谢、三羧酸循环及脂肪酸代谢等代谢通路变化与酒精诱导的急性肝损伤密切相关。且模型组大鼠尿液及肝组织中部分代谢物的变化趋势与CCl4诱导的急性肝损伤大鼠的相类似。4.分别采用ECF衍生尿液的GC/MS及TMS衍生血清的GC/TOFMS方法,对肝癌患者和正常对照人群的尿液及血液进行代谢组学研究,尝试为肝癌诊断以及肝癌的病理机制理解寻找新的切入点。研究结果发现,正常人与肝癌患者尿液和血液的代谢谱均有分离趋势,进一步利用监督的正交偏最小方差判别分析(OPLS-DA)方法能清晰地观察到肝癌患者与正常对照之间尿液和血液代谢轮廓的差异,通过对代谢网络变化的分析发现了与肝癌临床病理相关的代谢通路――能量代谢、苯丙氨酸、色氨酸、糖代谢以及一些其它的氨基酸代谢的变化。本课题在建立肝组织代谢组学方法的基础上,应用传统药理学指标与代谢组学研究所揭示的生物化学变化相结合的方法,为CCl4及酒精诱导的大鼠急性肝损伤的机制研究及药物药效作用的整体评价提供新的思路和视野;并且发现代谢组学技术在肝癌的早期诊断甚至分期上有很大的潜力,可以为临床诊断提供科学依据,非常值得进行深入一步的研究。更多还原

【Abstract】 The liver is the largest and most metabolically complex organ in humans. It plays a major role in metabolism and has a number of functions in the body, including glycogen storage, decomposition of red blood cells, plasma protein synthesis, and detoxification. So it is important to investigate the disorder of metabolisms in liver disease. Acute liver injury is the beginning of the liver diseases emerging, developing to the liver function failure at last. It is therefore of vital importance to investigate the pathogenesy and alternative measures of acute liver injury.It is well known that the mouse model of acute liver injury has been shown to be very useful for investigating the mechanism of various kinds of liver diseases and evaluating the drugs for hepatitis. Several animal models of liver injury, such as carbon tetrachloride (CCl4), alcoholic induced injury liver, have been widely used in both pathological and pharmacological studies. On the basis of the animal experiment, we try to find feasible method to research the mechanism of liver cancer from a new angle. Liver cancer, generally means primary liver cancer (PLC), is one of the most common malignant tumors of liver representing one of the most serious human cancerous problems in the world. PLC represents the fifth leading deaths among the cancer worldwide, and the second in China. As a multi-factorical and polygenic condition, PLC has complex molecular mechanisms which are not well understood by biomedical scientists and pathologists. Metabonomics as a novel methodology arising from the postgenomics era will help us to explore the mechanism study of liver injury and liver diseases.In this study, we try to establish extraction and chromatography in hyphenation with mass spectrometry metabonomic technologies to investigate metabolic variations of the liver tissue samples. Meanwhile, we investigate metabolic variations in PLC patients, alcoholic-induced acute liver injury rats, CCl4-induced acute liver injury rats and the protective effect of diammonium glycyrrihizinate on CCl4-induced liver injury rats using metabonomic study.We have developed a simple and reliable analytical assay for low-molecular-weight metabolites in liver tissue samples. The ethyl chloroformate (ECF) derivatization, GC/MS-based and the trimethylsilylation (TMS) derivatization, Gas chromatography time-of-flight mass spectrometry (GC/TOFMS)-based liver tissue metabonomic analysis method was optimized, respectively. Perchloric acid and ethonal extraction of liver tissue sample is suit to the GC/MS analysis and chloroform/methanol/water system is in favour of GC/TOFMS analysis. The two method validations revealed a wide linearity range, good repeatability and acceptable recovery rate for the proposed method.Combination with biochemistry and histological research, we used ECF derivatization and GC/MS-based metabonomic analysis of urines and liver tissue samples to visualize significant alterations in metabolite expression patterns as a result of CCl4-induced metabolic responses. The in-depth study of these metabolite alterations also allowed several major metabolic pathways, such as the tricarboxylic acid (TCA) cycle and some amino acid metabolism, were closely associated with CCl4 toxicity. Meanwhile, we attempt to investigate the protective actions of diammonium glycyrrihizinate to CCl4-induced liver injury with the metabonomic analysis. It will help make for the whole appraisal of the medicine.Establishing the acut alcoholic liver injury model of rats, we used ECF derivatization and GC/MS-based metabonomic technique to observe the metabolite expression pattern change in the urines and liver tissue samples. The results above could offer a feasible method to understand the mechanism of alcoholic liver injury and provide the basis for further research of alcoholic liver disease. The similar separation results were obtained with urine and liver tissue metabolic profiles. After identification of metabolites significantly varied in the model rats, energy metabolism, methionine metabolism and some other amino acids metabolism were associated with alcoholic-induced acute liver injury.Urinary and serumal metabolite profiling of PLC patients investigated using derivatized GC/MS in conjunction with modern multivariate statistical techniques revealed drastic biochemical changes as evidenced by fluctuations of metabolites. After identification of urine metabolites significantly varied in the PLC patients, energy metabolism, tyrosine metabolism were associated with PLC morbidity. The similar separation results of serum metabomomics were obtained with urine metabolic profiles, which were carried to indicate that the increased levels of histidine and glycine, and the decreased level of alanine, serine, lysine, phenylalanine and tryptophane.These results suggests that our metabonomics method may play an important role in exploring the mechanism of CCl4 or alcohol-induced liver injury, evaluating pharmacodynamic action of medicine for acute liver injury, and digging the great potential of urine or serum metabolic profile in PLC diagnosis. Meanwhile, the tissue metabonomics provide a unique perspective of localized metabolic information to metabonomics research.更多还原