【摘要】：目的：冠心病診治及預(yù)防亟需發(fā)掘新的靶點(diǎn)及方向。本研究旨在通過(guò)代謝組學(xué)技術(shù)篩選冠心病特異性代謝標(biāo)志物,并聯(lián)合宏基因組學(xué)技術(shù)尋找腸道菌群參與冠心病患者代謝的證據(jù)、特異性菌群及關(guān)聯(lián)代謝標(biāo)志物。方法：(1)通過(guò)采用高效液相色譜質(zhì)譜聯(lián)用技術(shù)對(duì)40名冠心病患者和43名健康對(duì)照者的血漿樣本進(jìn)行非靶向代謝組學(xué)研究,結(jié)合單變量統(tǒng)計(jì)和多變量統(tǒng)計(jì)方法篩選出可以明確區(qū)分冠心病患者與健康對(duì)照人群的代謝物、運(yùn)用聚類(lèi)分析方法區(qū)分不同表型下代謝物的變化情況,進(jìn)一步追溯這些代謝物的來(lái)源及所涉及的功能通路,找出冠心病患者特征性的代謝標(biāo)志物,從而建立研究冠心病患者代謝特征及代謝標(biāo)志物的分析方法。(2)通過(guò)采用上述方法,對(duì)另外59名冠心病患者和43名健康對(duì)照者的血漿和尿液進(jìn)行代謝分析,篩選出具有良好診斷能力的共同代謝標(biāo)志物,結(jié)合宏基因組學(xué)技術(shù)與腸道菌群的物種、功能水平進(jìn)行關(guān)聯(lián),發(fā)現(xiàn)與冠心病相關(guān)的特異性菌群。結(jié)果：(1)本實(shí)驗(yàn)通過(guò)高效液相色譜質(zhì)譜聯(lián)用技術(shù)對(duì)樣本進(jìn)行非靶向代謝組學(xué)研究,結(jié)合多種統(tǒng)計(jì)方法篩選具有良好診斷能力的代謝物,運(yùn)用聚類(lèi)分析等方法區(qū)分不同表型下代謝物變化情況,進(jìn)一步通過(guò)代謝物數(shù)據(jù)庫(kù)追溯代謝物來(lái)源和涉及的通路,最終聯(lián)合宏基因組學(xué)技術(shù)與腸道菌群進(jìn)行關(guān)聯(lián)分析,從而建立了冠心病代謝物研究及與菌群關(guān)聯(lián)分析的方法。(2)通過(guò)上述方法對(duì)40名冠心病患者和43名健康對(duì)照者的血漿樣本進(jìn)行分析,發(fā)現(xiàn)7種代謝物(1-萘酚、2-萘酚、甲基衣康酸、N-乙酰-D-葡萄糖胺-6-磷酸、L-肉毒堿、二甲酯馬來(lái)酸、苯丙酮酸)可以顯著區(qū)分冠心病患者和健康對(duì)照者,并發(fā)現(xiàn)N-乙酰-D-葡萄糖胺-6-磷酸和L-肉毒堿可能與腸道菌群參與的代謝相關(guān)。(3)對(duì)59名冠心病患者和43名健康對(duì)照者的血漿和尿液進(jìn)行代謝分析,篩選出具有良好診斷能力的共同代謝標(biāo)志物N-乙酰-D-葡萄糖胺-6-磷酸和甘露醇,并發(fā)現(xiàn)腸道菌群Clostridium sp. HGF2與N-乙酰-D-葡萄糖胺-6-磷酸、甘露醇顯著正相關(guān),而Streptococcus sp. M334 和 Streptococcus sp. M143則與甘露醇顯著正相關(guān)。結(jié)論：冠心病患者存在代謝紊亂,腸道菌群與其有關(guān),發(fā)現(xiàn)的代謝物和顯著相關(guān)的腸道菌群可能成為診斷冠心病的潛在標(biāo)志物和治療干預(yù)靶點(diǎn),另外本研究中所采用的研究方法或可為今后包括冠心病在內(nèi)心血管疾病代謝及腸道菌群研究提供借鑒。
[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.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R541.4

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