阳虚质粪便代谢组学特征及其与肠道菌群结构的相关性研究

发布时间：2018-08-30 17:59

【摘要】：目的:采用基于核磁共振(NMR)的代谢组学方法,探寻阳虚体质与平和体质分类的客观依据标准及微观物质代谢基础。并通过R-3.3.2统计软件,运用因子分析(Psych)及典型相关分析(Stats)方法,将代谢组学结果与实验前期的菌群结构结果进行相关性分析。验证"阳虚质肠道菌群结构的变化影响其代谢功能,并存在与代谢相关的特征功能菌群"的推论。方法:纳入符合体质分型判定标准者共60例,按体质类型分为:阳虚体质组30例,平和体质组30例。采集研究对象的粪便样品,运用1H-NMR技术和多元统计学分析方法研究不同体质者粪便的代谢组学特征,确定其潜在生物标记物。并寻找与阳虚质典型代谢标志物相关且影响宿主共代谢的特征功能菌群。结果:对比分析阳虚体质者与平和体质者粪便的典型代谢产物谱特征,以平和质者作为比较对象,结果显示:1.将代谢组学技术作为体质判别的客观理论依据是现实可行的。与平和质相比,代谢通路的改变切实存在于阳虚体质者中。2.发现阳虚体质者及平和体质者的潜在特征生物代谢标志物,并确定其浓度的变化趋向。两种体质中显著的潜在标记物有:乳酸(Lactate,δ1.33-1.35)、丙氨酸(Alanine,δ1.46-1.50)、丙酸盐(Propionate,81.05-1.07)、醋酸盐(Acetate,81.90-1.93)、丁酸盐(Butyrat e,80.88-0.92,1.56,1.57)、琥珀酸盐(Succinate,δ2.41)、蛋氨酸(Propionate/Methionine,δ2.16-2.19)、葡萄糖(Glucose/AA,δ3-4)。其中阳虚质组粪便中乳酸(Lactate,δ1.33-1.35)、丙氨酸(Alanine,81.46-1.50)、丙酸盐(Propionate,δ1.05-1.07)的浓度较平和质组高;醋酸盐(Acetate,δ1.90-1.93)、丁酸盐(Butyr ate,δ0.88-0.92,1.56,1.57)、琥珀酸盐(Succinate,δ2.41)、蛋氨酸(Propiona te/Methionine,82.16-2.19)、葡萄糖(Glucose/AA,δ3-4)的浓度较平和质组低。两种体质中较明显的潜在标记物有:缬氨酸(Ileucine/Leucine/Valine,80.95-0.97)、亮氨酸(Lysine/Leucine/Arginine,81.71-1.79)、谷氨酸盐(Glutamate,82.05,2.06)、赖氨酸(Lysine,δ3.01-3.04)、TMA(δ2.94,2.95)、精氨酸(Arginine,δ3.25-3.27)、甘氨酸(Glycine,δ3.57)、β 葡萄糖(β-glucose,δ4.65)、α 葡萄糖(α-glucose,δ5.24,5.25)。其中阳虚质组粪便中缬氨酸(Ileucine/Leucine/Vali ne,δ0.95-0.97)、亮氨酸(Lysine/Leucine/Arginine,δ1.71-1.79)、谷氨酸盐(G lutamate,δ2.05,2.06)、赖氨酸(Lysine,δ3.01-3.04)的浓度较平和体质组较高;TMA(δ2.94,2.95)、精氨酸(Arginine,δ3.25-3.27)、甘氨酸(Glycine,δ3.57)、β葡萄糖(β-glucose,δ4.65)、α葡萄糖(α-glucose,δ5.24,5.25)的浓度较平和体质组较低。3.通过比对相关数据库联合查阅文献,阐明了具有明显差异的代谢物的生理功能和代谢途径。发现阳虚体质者与平和体质者在氨基酸代谢、能量代谢、糖代谢以及神经递质、脏腑的生理功能变更等各生物生化方面具有其独特的个性差异,为阳虚体质人群的客观化分类提供了清晰准确的代谢组学支持。4.通过多变量统计学因子关联分析,寻找到与阳虚质典型代谢标志物相关且影响宿主共代谢的特征功能菌群:阳虚质中含量偏高的明串珠菌属(Leuconostoc)、罗氏菌属(Rothia)与α葡萄糖、丁酸盐、乳酸、丙氨酸、醋酸盐、谷氨酸盐、蛋氨酸、琥珀酸盐、TMA、赖氨酸、精氨酸、葡萄糖等代谢物相关,阳虚质中含量偏高的Defluviitaleaceae_incertae_sedis、Butyric imonas与α葡萄糖等代谢物相关;平和质中含量偏高的巨型球菌属(Megasphaera)与α葡萄糖等代谢物相关,平和质中含量偏高的毛螺菌属(Lachnospira)与葡萄糖、α葡萄糖、β葡萄糖等代谢物相关,平和质中含量偏高的瘤胃球菌属(Ruminococcus)与葡萄糖、α葡萄糖、β葡萄糖、丁酸盐、乳酸、丙氨酸、醋酸盐、谷氨酸盐、蛋氨酸、琥珀酸盐、TMA、赖氨酸、精氨酸等代谢物相关,这些启示可用于充分挖掘对菌群与各种复杂的影响因素之间的相关性的认知,具有深刻现实意义。结论:1.阳虚体质者与平和体质者在氨基酸代谢、能量代谢、糖代谢以及神经递质、脏腑的生理功能变更等各生物生化方面具有其独特的个性差异,为阳虚体质人群的客观化分类提供了清晰准确的代谢组学支持。两种体质的NMR代谢谱之间存在一定差异,相较于平和质,阳虚质代谢产物的典型综合特点为代谢标志物的含量降低,某些标志物出现应激性回馈性略微升高。特别是能量代谢、糖代谢以及氨基酸代谢的紊乱,应与相关脏腑的生理功能减退有关。为了理解肠道菌群与宿主代谢关系的分子基础,帮助促进宿主健康与更好地诊疗疾病,实验从代谢组学分析中找出特异的标志性代谢产物。2.获得与阳虚质典型代谢标志物相关且影响宿主共代谢的特征功能菌群,可初步验证"阳虚体质者肠道菌群结构的改变影响其宿主的代谢功能,并存在与代谢相关联的特征功能菌群"的推论。以明确肠道菌群中与宿主生理及健康相关的、影响宿主代谢的微观机制,促进了宿主健康并提供了诊疗保障。
[Abstract]:Objective: To explore the objective criteria and micro-material metabolic basis of classification of Yang-deficiency constitution and peaceful constitution by using nuclear magnetic resonance (NMR) based metabonomics, and to analyze the metabolomic results with the results of microbial community structure in the early stage of experiment by using R-3.3.2 statistical software, factor analysis (Psych) and canonical correlation analysis (Stats). Methods: 60 cases were included in the study. They were divided into two groups according to their constitution types: 30 cases of Yang deficiency constitution group and 30 cases of calm constitution group. NMR and multivariate statistical analysis were used to study the metabolomic characteristics of feces in different constitutions, identify potential biomarkers, and search for functional microflora associated with typical metabolic markers of Yang deficiency and affecting co-metabolism of host. Compared with the peaceful constitution, the changes of metabolic pathway actually exist in the patients with Yang-deficiency constitution. 2. The potential characteristics of metabolic markers in the patients with Yang-deficiency constitution and those with peaceful constitution were found and their concentrations were determined. Significant potential markers in both constitutions are lactic acid (Lactate, Delta 1.33-1.35), alanine (Alanine, Delta 1.46-1.50), propionate (Propionate, 81.05-1.07), acetate (Acetate, 81.90-1.93), butyrate (Butyrat, 80.88-0.92, 1.56, 1.57), succinate (Succinate, Delta 2.41), methionine (Propiate/Methionate, Delta 2.16-2.16-2.41). The concentrations of lactic acid (Lactate, Delta 1.33-1.35), alanine (Alanine, 81.46-1.50), propionate (Propion ate, Delta 1.05-1.07), acetate (Acetate, Delta 1.90-1.93), butyrate (Butyr ate, Delta 0.88-0.92, 1.56, 1.57), succinate (Succinate, Delta 2.41), methionine (Propion ate) in the feces of the Yang deficiency group were higher than those of the normal group. A te/Methionine, 82.16-2.19, and glucose (Glucose/AA, Delta 3-4) concentrations were lower than those in the plain group. The more obvious potential markers in the two constitutions were valine (Ileucine/Leucine/Valine, 80.95-0.97), leucine (Lysine/Leucine/Arginine, 81.71-1.79), glutamate (Glutamate, 82.05, 2.06), lysine (Lutamate, Delta 3.01-3.04), TMA (delta 2.94, TM 2.94). 95, arginine (Arginine, Delta 3.25-3.27), glycine (Glycine, Delta 3.57), beta glucose (beta-glucose, Delta 4.65), alpha glucose (alpha-glucose, Delta 5.24, 5.25). Among them, valine (Ileucine/Leucine/Vali ne, Delta 0.95-0.97), leucine (Lysine/Leucine/Arginine, Delta 1.71-1.79), glutamate (G-lutte, Delta 2.05, 2.06), lysine (Lysine/Leucine/Arginine, Delta 1.71-1.79), glutamate (G-lutte, Delta 2.05, 2. (Lysine, Delta 3.01-3.04) had a higher concentration than that of the calm group; TMA (delta 2.94, 2.95), arginine (Arginine, Delta 3.25-3.27), glycine (Glycine, Delta 3.57), beta glucose (beta-glucose, Delta 4.65), and alpha glucose (a-glucose, Delta 5.24, 5.25) had a lower concentration than that of the calm group.3. It was found that there were distinct individual differences in amino acid metabolism, energy metabolism, glucose metabolism, neurotransmitter, physiological function of viscera between the two groups, which provided clear and accurate metabolism for the objective classification of the people with Yang deficiency constitution. Histological support. 4. Through multivariate statistical factor correlation analysis, we found the characteristic functional microflora related to the typical metabolic markers of Yang deficiency and affecting the co-metabolism of host: Leuconostoc, Rothia and alpha glucose, butyrate, lactic acid, alanine, acetate, glutamate, methionine with high content of Yang deficiency. Defluviita leaceae_incertae_sedis and Butyric imonas with high content of Yang deficiency substance are related to metabolites such as acid, succinate, TMA, lysine, arginine and glucose; Megasphaera with high content of neutral substance is related to metabolites such as alpha-glucose, and snail with high content of neutral substance is related to Defluviita leaceae_incertae_sedis and Butyric imonas with high content of alpha-glucose. Lachnospira is associated with glucose, alpha-glucose, beta-glucose and other metabolites. Ruminococcus, which is high in the serum, is associated with glucose, alpha-glucose, beta-glucose, butyrate, lactic acid, alanine, acetate, glutamate, methionine, succinate, TMA, lysine, arginine and other metabolites. These enlightenments may include It has profound practical significance to fully tap the cognition of the correlation between the flora and various complex influencing factors. Conclusion: 1. The Yang-deficiency constitution and the peaceful constitution have their unique personality differences in amino acid metabolism, energy metabolism, glucose metabolism, neurotransmitters, physiological function changes of viscera, etc. The objective classification of Yang-deficiency constitution provides clear and accurate metabonomic support. There are some differences between the NMR metabolic profiles of the two constitutions. Compared with the placid constitution, the typical comprehensive characteristics of the metabolic products of Yang-deficiency constitution are that the content of metabolic markers decreases, and some markers show slightly increased stress feedback. In order to understand the molecular basis of the relationship between intestinal flora and host metabolism, and to help promote host health and better diagnosis and treatment of diseases, the specific metabolite markers were identified from metabolomic analysis. 2. Typical metabolic markers of Yang deficiency were obtained. The characteristic functional microflora related to the host and affecting the host's co-metabolism can preliminarily verify the inference that the alteration of intestinal flora structure in the patients with Yang deficiency constitution affects the host's metabolic function and there are characteristic functional microflora associated with metabolism. It promotes the health of the host and provides medical support.
【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R229

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