【作者】 张红栓；

【导师】 贾钰华；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 研究背景：作为目前我国国民首位致死原因的心血管疾病是一种与代谢过程密切相关的复杂疾病,发病率和死亡率依然呈上升趋势。其发生、发展与机体在代谢过程中引起的体液和组织中代谢产物的变异及表达异常有关。辨证论治是中医学的主要特色,整个诊断、治疗、处方、用药都是以证为核心展开的。中医学诊治冠心病的切入点即证候。代谢组学是继基因组学、蛋白质组学等之后发展起来的以探讨机体整体代谢状态为目标的新方法。疾病引起的机体代谢水平紊乱往往会在尿液和血液等体液的代谢组得到表现。对尿液和血液等体液代谢组进行检测和分析,就有可能对疾病发生和发展过程伴随的生物化学变化进行了解和认识,有可能发现相关疾病发生的早期代谢组标志物簇并认识相关的病理发生的分子机理,有可能对疾病在其早期,甚至发生之前进行诊断。研究目的：本研究利用代谢组学技术对冠心病心绞痛痰浊证、血瘀证的血液、尿液对比分析和气滞血瘀证、心血瘀阻证、气虚血瘀证三种血瘀证进行了初步研究,试图通过患者血液、尿液代谢物质的变化探索病证模式背后的生物标记物,为冠心病心绞痛痰浊证和血.瘀证本质的系统研究提供科学依据。建立病症结合的代谢组学研究技术平台,为从代谢物水平探讨中医证本质提供思路和方法。研究方法：1、冠心病心绞痛痰浊证、血瘀证患者的血浆代谢组学研究：对10例冠心病心绞痛痰浊证患者和10例冠心病心绞痛血瘀证患者的血浆样本进行氢核磁共振(1H NMR)检测。通过偏最小二乘法判别分析(PLS-DA)方法研究冠心病心绞痛痰浊证患者与血瘀证患者之间血浆代谢产物谱的差异。2、冠心病心绞痛痰浊证、血瘀证的尿液代谢组学研究：对10例冠心病心绞痛痰浊证患者和10例冠心病心绞痛血瘀证患者的尿液样本进行氢核磁共振(1H NMR)检测。通过偏最小二乘法判别分析(PLS-DA)研究冠心病心绞痛痰浊证患者与血瘀证患者之间尿液代谢产物谱的差异。3、冠心病心绞痛三种血瘀证的血浆代谢组学研究：对18例健康人、18例冠心病心绞痛血瘀证患者(6例气滞血瘀证、6例心血瘀阻证、6例气虚血瘀证)的血浆样本进行氢核磁共振(1H NMR)检测。通过偏最小二乘判别分析(PLS-DA)方法研究各组之间的血浆代谢产物谱差异。结果：冠心病心绞痛痰浊证患者与冠心病心绞痛血瘀证患者血清能够被区分,二组血浆代谢物含量存在明显差异。痰浊证组血浆中低密度脂蛋白、极低密度脂蛋白、脂类化合物、酮体、乳酸、葡萄糖、N-乙酰糖蛋白、门冬氨酸、异亮氨酸、谷氨酞胺、肌醇的含量高于血瘀证组,而高密度脂蛋白与不饱和脂肪酸、丙氨酸、瓜氨酸、精氨酸、脯氨酸的含量低于血瘀证组。冠心病心绞痛痰浊证患者与冠心病心绞痛血瘀证患者两组尿液核磁共振氢谱的PLS-DA结果显示,冠心病心绞痛痰浊证患者与冠心病心绞痛血瘀证患者能够被区分,二组尿液样本之间的代谢物含量存在明显差异。冠心病心绞痛痰浊证组尿液中柠檬酸、α-酮戊二酸、顺式-乌头酸、葡萄糖、3-羟基丁酸、丙酮、酪氨酸、肌酐、氧化三甲胺、二甲胺、马尿酸的含量高于血瘀证组,而胆汁酸、组氨酸的含量低于血瘀证组。冠心病心绞痛三种血瘀证的血浆代谢组学的PLS-DA结果示,与健康人相比,心绞痛血瘀证患者血浆柠檬酸、琥珀酸、葡萄糖、乙酰糖蛋白、低密度脂蛋白、极低密度脂蛋白、3-羟基丁酸、谷氨酸、脯氨酸、亮氨酸含量较高,高密度脂蛋白、不饱和脂肪酸、苏氨酸、组氨酸含量较低。气虚血瘀证组与其他两个血瘀证组差别较大,而气滞血瘀证与心血瘀阻证差别不明显。与其他血瘀证相比,气虚血瘀证组血浆柠檬酸、3-羟基丁酸、丙酮、氧化三甲胺含量较高,葡萄糖、N-乙酰糖蛋白、酪氨酸、苯丙氨酸、脯氨酸、亮氨酸含量较低。结论：各分组血液、尿液样本的1H NMR代谢谱存在差异,能从代谢组学分析中找出特异的标志性代谢产物。利用代谢组学方法能区分不同证型的冠心病心绞痛患者的血浆1H-NMR图谱,能确定痰浊证、血瘀证之间存在明显差异的代谢产物。尿液代谢物变化能在一定程度上区分冠心病心绞痛的不同中医证型。代谢组学研究的优势在于样品获取更加方便、检测更加容易。它能够对检测到的小分子化合物进行定性和定量研究,而这些化合物的上调、下调或许将来可以用于中医证候鉴别的客观指标。祖国医学和代谢组学均是对影响机体整体状况的多因素的研究对机体某一病理变化的全面情况进行探究。二者之间理论上的关联以及本研究的结果,为中医辨证分型客观化研究提供了新思路,为揭示中医学证的本质提供了新方法。健康人与冠心病心绞痛血瘀证患者的血浆1H-NMR图谱可以区分,能确定差异代谢物；心绞痛三种血瘀证中,气虚血瘀证组与其他两个血瘀证组血浆1H-NMR图谱差别较大,能确定差异代谢物,而气滞血瘀证与心血瘀阻证差别不明显。这为我们进一步研究冠心病证候本质提供了思路。更多还原

【Abstract】 Background:As the first main cause of death in our country, cardiovascular disease is a complex process closely related to metabolic, with morbidity and mortality increasing. Its occurrence, development associated with metabolite abnormalities in metabolism of body fluids and tissues. Syndrome identification is the key feature of Chinese medicine. The diagnosis, treatment, prescription, medication are begin at syndromes. The gate of medical diagnosis and treatment of coronary heart disease is Chinese Traditional Medicine syndromes. Metabolomics is a new method developed follow with genomics, proteomics to explore the overall metabolic state of the body. The body metabolism disorder caused by disease often show the performance by the metabolic group in body fluids such as urine and blood. Testing and analysis on the metabolism of body fluids such as urine and blood group will contribute: understanding to the biochemical changes accompanying the process of the development of the disease, the disclosure of the early metabolomics markers related diseases and recognization of the molecular mechanism of pathogenesis, and the diagnosis of disease in early stage. Purpose:In this study, metabonomics technology was used for comparison between blood, urine of angina pectoris patients with phlegm-turbid syndrome and blood-stasis syndrome, and among three kinds of blood-stasis syndrome:Qi-stagnation and blood-stasis syndrome (QSBS), heart blood-stasis syndrome (HBS), Qi-deficieney and blood-stasis syndrome (QDBS). We tried to explore syndrome biomarkers underlying patterns of disease by detecting changes of blood and urine metabolites and to provide a scientific basis for angina pectoris phlegm-turbid syndrome and blood-stasis syndrome study. We tried to apply ideas and methods to explore the nature of Chinese Traditional Medicine syndromes, by the establishment of disease and syndrome combining metabolomics research platforms.Methods:1, Plasma metabonomics study for angina pectoris patients with phlegm-turbid syndrome and blood-stasis syndrome:plasma samples from 10 patients with angina pectoris due to coronary heart disease phlegm-turbid syndrome patients and 10 patients with angina pectoris due to coronary heart disease blood-stasis syndrome were detected by nuclear magnetic resonance (’H NMR). Plasma metabolites profilling difference between angina pectoris patients due to coronary heart disease with phlegm-turbid syndrome or with blood-stasis syndrome was analysed by discriminant analysis (PLS-DA) method.2, Urine metabolomics study on angina pectoris patients due to coronary heart disease with phlegm-turbid syndrome, blood-stasis syndrome:urine samples from 12 patients with angina pectoris due to coronary heart disease phlegm-turbid syndrome patients and 12 patients with angina pectoris due to coronary heart disease blood-stasis syndrome were detected by nuclear magnetic resonance (’H NMR). Urine metabolites profilling difference between angina pectoris patients due to coronary heart disease with phlegm-turbid syndrome or with blood-stasis syndrome was analysed by discriminant analysis (PLS-DA) method.3, Plasma metabonomics study for angina pectoris patients due to coronary heart disease with three blood-stasis syndrome:plasma samples from 18 healthy people and 18 patients with angina pectoris due to coronary heart disease with three blood-stasis syndrome (6 cases QSBS,6例HBS,6 cases QDBS) were detected by nuclear magnetic resonance (1H NMR). Plasma metabolites profilling difference between healthy people and angina pectoris patients due to coronary heart disease with three blood-stasis syndrome was analysed by discriminant analysis (PLS-DA) method.Results:The plasma metabolite levels of angina pectoris patients due to coronary heart disease with phlegm-turbid syndrome and blood-stasis syndrome varied significantly. Compared with APPS patients, LDL, VLDL, Lipid,3-hydroxy-butyric acid, Acetone, Lactate, glucose, NAC, aspartate, isoleucine, glutamine and myc-inositol increased in APBS patients, while HDL, unsaturated lipid, Alanine, Citrulline, Arginine, Proline decreased considerably.PLS-DA of urine 1H NMR spectra revealed revealed different metabolic spectra between APPS patients and APBS patients, demonstrated that the metabolic characteristics of the two groups were significantly different. Compared with APPS patients, Citric acid,α-ketoglutarate, cis - aconitic acid, glucose,3 - hydroxybutyric acid, acetone, tyrosine, creatinine, trimethylamine oxide, dimethylamine, hippuric in urine of APBS patients increased clearly, while the bile acid, histidine decreased considerably.PLS-DA of plasma 1H NMR spectra revealed different metabolic spectra between healthy people and APBS patients. Compared with healthy people, citrate, succinate, glucose, glycoprotein, LDL, VLDL,3-hydroxybutyrate, glutamine, proline, leucine increased in APBS patients, while HDL, unsaturated lipid, threonine, histidine decreased considerably. Different metabolic spectra also were revealed between QDBS patients and other APBS patients. Compared with QSBS patients and HBS patients, Citrate,3-hydroxybutyrate, acetone, TMAO increased in QDBS patients, while glucose, NAC, tyrosine, phenylalanine, proline, leucine decreased.Conclusion:Characteristic metabolic products in each groups can be identified by 1H NMR based metabonomics analysis. Metabonomic study is a feasible and promising way to detect the variation of plasma and urine metabonomics of Angina Pectoris patients with different TCM Syndromes. Changes in urinary metabolites can distinguish different Chinese Traditional Medicine syndromes of angina pectoris patients due to coronary heart disease to some extent. The advantage of metabolomics is more convenient to obtain samples, and more easier to test. It can detect small molecule compounds for qualitative and quantitative research, and the up- and down-regulation of these which perhaps in the future can be used in identification of Chinese Traditional Medicine syndromes. Chinese medicine and metabolomics both explore the multi-body factors affecting the overall condition of a pathological change. The relationship between their theory and the results of this study provide a new idea of objective research to TCM Syndrome, and a new method to reveal the nature of TCM Syndrome.1H-NMR pattern of plasma between healthy people and patients with angina pectoris due to coronary heart disease with blood-stasis syndrome can be distinguished, and further determine the difference in metabolite; in three types of angina with blood-stasis syndrome, plasma’H-NMR pattern of QDBS vary greatly to the other groups, which can determine the difference in metabolites. Wherever there is no obvious differences between QSBS and HBS. This provides us a guideline to further explore the nature of Chinese Traditional Medicine syndromes of coronary.更多还原