基于HPLC-MS冠心病代谢组学及其与肠道菌群宏基因组学关联的研究

发布时间：2018-11-21 10:48

【摘要】：目的：冠心病诊治及预防亟需发掘新的靶点及方向。本研究旨在通过代谢组学技术筛选冠心病特异性代谢标志物,并联合宏基因组学技术寻找肠道菌群参与冠心病患者代谢的证据、特异性菌群及关联代谢标志物。方法：(1)通过采用高效液相色谱质谱联用技术对40名冠心病患者和43名健康对照者的血浆样本进行非靶向代谢组学研究,结合单变量统计和多变量统计方法筛选出可以明确区分冠心病患者与健康对照人群的代谢物、运用聚类分析方法区分不同表型下代谢物的变化情况,进一步追溯这些代谢物的来源及所涉及的功能通路,找出冠心病患者特征性的代谢标志物,从而建立研究冠心病患者代谢特征及代谢标志物的分析方法。(2)通过采用上述方法,对另外59名冠心病患者和43名健康对照者的血浆和尿液进行代谢分析,筛选出具有良好诊断能力的共同代谢标志物,结合宏基因组学技术与肠道菌群的物种、功能水平进行关联,发现与冠心病相关的特异性菌群。结果：(1)本实验通过高效液相色谱质谱联用技术对样本进行非靶向代谢组学研究,结合多种统计方法筛选具有良好诊断能力的代谢物,运用聚类分析等方法区分不同表型下代谢物变化情况,进一步通过代谢物数据库追溯代谢物来源和涉及的通路,最终联合宏基因组学技术与肠道菌群进行关联分析,从而建立了冠心病代谢物研究及与菌群关联分析的方法。(2)通过上述方法对40名冠心病患者和43名健康对照者的血浆样本进行分析,发现7种代谢物(1-萘酚、2-萘酚、甲基衣康酸、N-乙酰-D-葡萄糖胺-6-磷酸、L-肉毒碱、二甲酯马来酸、苯丙酮酸)可以显著区分冠心病患者和健康对照者,并发现N-乙酰-D-葡萄糖胺-6-磷酸和L-肉毒碱可能与肠道菌群参与的代谢相关。(3)对59名冠心病患者和43名健康对照者的血浆和尿液进行代谢分析,筛选出具有良好诊断能力的共同代谢标志物N-乙酰-D-葡萄糖胺-6-磷酸和甘露醇,并发现肠道菌群Clostridium sp. HGF2与N-乙酰-D-葡萄糖胺-6-磷酸、甘露醇显著正相关,而Streptococcus sp. M334 和 Streptococcus sp. M143则与甘露醇显著正相关。结论：冠心病患者存在代谢紊乱,肠道菌群与其有关,发现的代谢物和显著相关的肠道菌群可能成为诊断冠心病的潜在标志物和治疗干预靶点,另外本研究中所采用的研究方法或可为今后包括冠心病在内心血管疾病代谢及肠道菌群研究提供借鉴。
[Abstract]:Objective: to explore new targets and directions for diagnosis, treatment and prevention of coronary heart disease. The purpose of this study was to screen the specific metabolic markers of coronary heart disease (CHD) by metabonomics, and to search for evidence, specific flora and related metabolic markers of intestinal flora involved in the metabolism of patients with coronary heart disease (CHD) combined with macrogenomics. Methods: (1) the plasma samples of 40 patients with coronary heart disease and 43 healthy controls were studied by high performance liquid chromatography-mass spectrometry (HPLC / MS). In combination with univariate and multivariate statistical methods, the metabolites of patients with coronary heart disease and healthy controls could be clearly distinguished, and the changes of metabolites under different phenotypes were distinguished by cluster analysis. To further trace the sources of these metabolites and the functional pathways involved, and to identify characteristic metabolic markers in patients with coronary heart disease, Therefore, the metabolic characteristics and metabolic markers of coronary heart disease patients were studied. (2) the plasma and urine metabolism of 59 patients with coronary heart disease and 43 healthy controls were analyzed by using the above method. Screening out common metabolic markers with good diagnostic ability, combining with macrogenomics to correlate with the species and functional level of intestinal flora, and find specific flora related to coronary heart disease. Results: (1) the non-target metabolites of the samples were studied by high performance liquid chromatography-mass spectrometry (HPLC / MS), and the metabolites with good diagnostic ability were screened in combination with various statistical methods. Cluster analysis was used to distinguish the changes of metabolites under different phenotypes, and the metabolic sources and pathways were traced back through metabolites database. Finally, macrogenomics was combined with microflora to analyze the relationship between metabolites and intestinal flora. Therefore, a method for the study of metabolites in coronary heart disease and its association with bacterial flora was established. (2) the plasma samples of 40 patients with coronary heart disease and 43 healthy controls were analyzed and 7 metabolites (1-naphthol, 1-naphthol) were found. 2-naphthol, methyl-itaconic acid, N-acetyl-D-glucosamine 6-phosphoric acid, L-carnitine, dimethyl maleic acid, phenylpyruvate) significantly distinguished coronary heart disease patients from healthy controls. It was found that N-acetyl-D-glucosamine 6-phosphoric acid and L-carnitine might be related to the metabolism of intestinal flora. (3) the plasma and urine metabolism of 59 patients with coronary heart disease and 43 healthy controls were analyzed. A common metabolic marker, N-acetyl-D-glucosamine 6-phosphoric acid and mannitol, with good diagnostic ability was screened out, and Clostridium sp. was found in intestinal flora. HGF2 was positively correlated with N-acetyl-D-glucosamine 6-phosphoric acid and mannitol, while Streptococcus sp. was positively correlated with N- acetyl-D-glucosamine-6-phosphoric acid and mannitol. M334 and Streptococcus sp. M143 was positively correlated with mannitol. Conclusion: there is metabolic disorder in patients with coronary heart disease, and the intestinal flora is related to it. The metabolites and the significantly related intestinal flora may be potential markers and therapeutic intervention targets for the diagnosis of coronary heart disease. In addition, the research methods used in this study may provide references for future studies on metabolism of cardiovascular diseases, including coronary heart disease, and intestinal flora.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R541.4

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