GC-MS技术对儿童紫癜性肾炎热伤肾络证与肾阴不足证的分析研究

发布时间：2018-01-28 21:47

本文关键词： 气相色谱质谱联用技术紫癜性肾炎热伤肾络证肾阴不足证尿液代谢物　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:通过代谢组学气相色谱-质谱联用技术(GC-MS)研究儿童紫癜性肾炎热伤肾络证与肾阴不足证尿液样本代谢物,比较两种证型物质基础,寻找紫癜性肾炎实证与虚证差异性代谢产物,对差异性代谢产物进行通路分析,探求儿童紫癜性肾炎中医虚实证候可能存在的客观性指标。方法:采用气相色谱-三重四级杆质谱仪检测儿童紫癜性肾炎热伤肾络证组(21例)、肾阴不足证组(19例)及正常健康儿童组(30例)尿液代谢产物,运用XCMS在线数据处理,获得化合物的相对峰面积,使用主成分分析(PCA)及正交偏最小二乘法-判别分析(OPLS-DA)对样本数据进行多维统计学分析,筛选出各组潜在生物标记物,运用MetaboAnalyst分析可能涉及的相关代谢通路综合评价,阐释儿童紫癜性肾炎证候分型的物质基础及可能的机制。结果:1.儿童紫癜性肾炎两证型组与正常组在OPLS-DA中三组可以区分,其中OPLS-DA模型参数为 R2Y=0.802,Q2=0.205。2.紫癜性肾炎热伤肾络证组与正常组在OPLS-DA中可以明显区分,模型参数为R2Y=0.922,Q2=0.342,根据VIP值大于1,筛选出14种内源性化合物,分别为α-羟基异丁酸、羟基乙酸、丙酸、胱硫醚、L-苏糖醇、赤藓糖醇、L-丙氨酸、戊烯二酸、柠檬酸、3-(3-羟基苯基)-3-羟基丙酸、D-甘露醇、D-葡糖酸、N-α-乙酰-L-赖氨酸、鲨肌醇。3.紫癜性肾炎肾阴不足证组与正常组在OPLS-DA中可明显区分,模型参数为R2Y=0.985,Q2=0.553,最终筛选出肌酐、1-氨基环丙烷羧酸、牛磺酸、棕榈酸、葡糖酸、马尿酸、硬脂酸、纤维二糖、鲨肌醇9种内源性代谢产物。4.紫癜性肾炎热伤肾络证与肾阴不足证两组在OPLS-DA中可明显区分,模型参数为R2Y=0.972,Q2=0.566,最终鉴定出4种内源性化合物,分别为:柠檬酸、6-氮胸腺嘧啶、纤维二糖、N-α-乙酰-L-赖氨酸。讨论:1.与正常组比较,牛磺酸在肾阴不足证组中呈下调趋势;胱硫醚在热伤肾络证组呈上调趋势;赖氨酸在热伤肾络证组呈下调趋势;鲨肌醇在热伤肾络证组及肾阴不足证组均呈上调趋势;柠檬酸在热伤肾络证组中呈下调趋势。2.与热伤肾络证组比较,赖氨酸、柠檬酸、6-氮胸腺嘧啶、纤维二糖在肾阴不足证中呈上调趋势。3.对所有代谢产物进行代谢通路分析,发现儿童紫癜性肾炎主要涉及的代谢通路为:氮代谢,半胱氨酸和蛋氨酸代谢,牛磺酸和亚牛磺酸代谢,戊糖磷酸途径,柠檬酸循环(TCA循环),赖氨酸降解。上述结果表明在儿童紫癜性肾炎尿液代谢产物与正常组可以明显区分,热伤肾络证与肾阴不足证具有差异性物质基础,差异化合物主要涉及氨基酸代谢及能量代谢异常。
[Abstract]:Objective: to study the metabolites in urine samples of children with purpura nephritis with heat injury and deficiency of kidney yin by gas chromatography-mass spectrometry (GC-MS), and to compare the substance basis of the two syndrome types. To search for the differential metabolites of purpura nephritis and deficiency syndrome, and analyze the pathway of different metabolites. Objective index of deficiency and excess syndrome of TCM in children with Henoch-Schonlein purpura nephritis (HSPN). Methods: 21 cases of children with Henoch-Schonlein purpura nephritis were detected by gas chromatography-four-step mass spectrometer. Urine metabolites in 19 cases of deficiency of kidney yin syndrome and 30 cases of healthy children) were processed with XCMS online data to obtain the relative peak area of the compounds. Principal component Analysis (PCA) and orthogonal partial least Square-discriminant Analysis (OPLS-DA) were used to carry out multidimensional statistical analysis of the sample data and to screen out potential biomarkers in each group. Comprehensive evaluation of related metabolic pathways may be involved by MetaboAnalyst analysis. To explain the material basis and possible mechanism of syndrome classification of Henoch-Schonlein purpura nephritis in children. Results 1. In OPLS-DA, the two syndrome types of children with purpura nephritis can be distinguished from the normal group. The parameters of OPLS-DA model were R2YP0.802Q2O0.205.2.The renal collateral syndrome of heat injury of purpura nephritis could be distinguished from that of normal group in OPLS-DA. The model parameters were R2YP0. 922OQ2O0.342. According to the VIP value greater than 1, 14 endogenous compounds were selected, which were 伪 -hydroxyisobutyric acid, glycolic acid, propionic acid and cystathion. L-threonol, erythritol, L-alanine, pentenedioic acid, citric acid, 3-hydroxy-3-hydroxyphenyl -3-hydroxypropionic acid, D-mannitol, D-glucoic acid, N- 伪 -acetyl-L-lysine. Shark inositol .3.The model parameter is R2YT 0.985Q2O0.553. Creatinine was screened out in the model group and the normal group and the group with deficiency of kidney yin in Henoch-Schonlein purpura nephritis were distinguished obviously in OPLS-DA. 1-aminocyclopropane carboxylic acid, taurine, palmitic acid, glucosinic acid, hippuric acid, stearic acid, fibronectose. Shark inositol 9 kinds of endogenous metabolites .4. the two groups of syndrome of kidney meridian injury and deficiency of kidney yin can be distinguished in OPLS-DA, the model parameter is R2YN 0.972. Finally, four endogenous compounds were identified as citrate 6-azotouracil, fibronectin N- 伪 -acetyl-L-lysine. Taurine showed a downward trend in the group of deficiency of kidney yin. Cystathithione showed an upward trend in the heat injury kidney collaterals group; Lysine showed a down-regulation trend in the heat injury kidney collaterals syndrome group; Shark inositol showed an upward trend in heat injury and deficiency of kidney yin group. Citric acid in heat injury kidney collateral syndrome group showed a downward trend .2.Compared with heat injury kidney collateral syndrome group, lysine, citric acid 6-azotinopyrimidine. Analysis of metabolic pathway of all metabolites found that the main metabolic pathways involved in Henoch-Schonlein purpura nephritis in children were nitrogen metabolism, cysteine and methionine metabolism. Taurine and subtaurine metabolism, pentose phosphoric acid pathway, citric acid cycle TCA cycle, lysine degradation. Heat injury kidney collaterals syndrome and kidney yin deficiency syndrome have different material basis, the difference compounds mainly involve amino acid metabolism and energy metabolism abnormality.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R272

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