基于~1H NMR技术的奶牛Ⅰ型和Ⅱ型酮病血浆代谢组学分析

发布时间：2018-03-25 17:11

本文选题：Ⅱ型酮病　切入点：Ⅰ型酮病　出处：《黑龙江八一农垦大学》2015年硕士论文

【摘要】：酮病是围产期高产奶牛常见而多发的一种能量代谢障碍性疾病。本病主要以低血糖、高酮体和高游离脂肪酸为生化特征；能量负平衡（Negative energy balancen, NEB）、脂肪过度动员为本病的病理学基础。通常根据血中酮体水平及临床症状，奶牛酮病被分为临床型酮病以及亚临床型酮病，有关它们的病因学研究已有很多。然而，借鉴于糖尿病的分类，根据血中酮体、葡萄糖以及游离脂肪酸的水平，奶牛酮病又被分为Ⅰ型酮病和Ⅱ型酮病。目前，关于奶牛Ⅱ型酮病的代谢组学研究报道极少。因此，本研究应用核磁共振技术并结合多元统计分析，对Ⅰ型酮病和Ⅱ型酮病的奶牛血浆进行全景式代谢变化的研究，旨在筛选出奶牛Ⅰ型酮病和Ⅱ型酮病的差异性特征性代谢物，为丰富奶牛Ⅱ型酮病的病因学以及奶牛酮病防治新策略提供科学依据。本研究根据血浆中β-羟丁酸（BHBA）、葡萄糖（Glc）和游离脂肪酸（NEFA）的水平，选取了50头产后7-28天试验奶牛，其中无其他疾病的Ⅰ型酮病组奶牛（K1，n=20头）血浆中BHBA1.20mmol/L，Glc2.50mmol/L，NEFA0.50mmol/L；Ⅱ型酮病组奶牛（K2，n=20头）血浆中BHBA1.20mmol/L，Glc2.80mmol/L, NEFA0.50mmol/L；健康对照组奶牛（C，n=10头）血浆中BHBA1.00mmol/L，Glc3.75mmol/L，NEFA0.40mmol/L。先运用核磁共振技术（1H Nuclear Magnetic Resonance，1H NMR）对三组试验奶牛的血浆样品进行代谢物检测，而后进行主成分分析（Principle ComponentAnalysis，PCA）、偏最小二乘方方法（Partial Least Squares-discriminant Analysis，PLS-DA）和正交偏最小二乘方方法（Orthogonal Partial Least-Squares DiscriminantAnalysis，OPLS-DA）等多元统计分析，再应用生物学信息技术对差异性代谢物进行生物学分析。最后采用酶联免疫（EnzymeLinked Immunosorbent Assays，ELISA）方法对Ⅰ型酮病和Ⅱ型酮病筛选出的内源特征性差异代谢物进行验证。 Ⅰ型酮病、Ⅱ型酮病与健康对照组奶牛三组间代谢成分存在显著差异。鉴定出26种差异代谢物，相对于健康对照，Ⅰ型酮病血浆中升高的2种物质为β-羟丁酸、丙酮；降低的15种代谢物为丙氨酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、赖氨酸、柠檬酸、肌酸、甲酸、脂质、肌醇、O-乙酰葡萄糖胺、磷酸胆碱、α-葡萄糖、β-葡萄糖，有显著的差异(P0.05)。相对于健康对照，Ⅱ型酮病血浆中升高的3种代谢物为β-羟丁酸、丙酮、乳酸；降低的4种代谢物为丙氨酸、赖氨酸、酪氨酸、肌酸，有显著的差异(P0.05)。与Ⅱ型酮病相比，Ⅰ型酮病血浆中升高的8种代谢物为β-羟丁酸、乙酸、丙酮、异亮氨酸、亮氨酸、缬氨酸、低密度脂蛋白和极低密度脂蛋白；降低的14种代谢物为丙氨酸、柠檬酸、甲酸、谷氨酸、谷氨酰胺、甘氨酸、组氨酸、脂质、赖氨酸、肌醇、O-乙酰葡萄糖胺、磷酸胆碱、α-葡萄糖、β-葡萄糖，有显著的差异(P0.05)。将差异性代谢物通过Kyoto Encyclopedia of Genes andGenomes（KEGG）数据库分析，检测到的代谢物不仅参与糖代谢、脂肪代谢和氨基酸代谢等能量代谢，也参与酮体的形成。从这些差异性代谢物中筛选出与奶牛Ⅰ型和Ⅱ型酮病代谢密切相关的11种差异性代谢物为丙酮酸、α-酮戊二酸、胆碱磷酸甘油酯、草酰乙酸、甘氨酸、酪氨酸、柠檬酸、异亮氨酸、亮氨酸、丙氨酸、乳酸，并用ELISA方法对这11种差异代谢物进行验证，其浓度变化结果与1H NMR技术的检测结果相符。应用代谢组学的1H NMR技术筛选和确证了Ⅰ型酮病和Ⅱ型酮病的血浆差异代谢物，不仅验证了奶牛酮病发生过程中糖、脂类和氨基酸的代谢紊乱，，也为今后进一步探究Ⅱ型酮病的发病机制和防治奠定理论基础。
[Abstract]:Ketosis is a kind of metabolic disorders in dairy cows perinatal common and frequently encountered disease. This is mainly to hypoglycemia, high ketone and high free fatty acids and biochemical characteristics; negative energy balance (Negative energy, balancen, NEB), excess fat mobilization for the pathology of this disease is usually based on the ketone base. The level of blood and clinical symptoms of ketosis were divided into subclinical ketosis and subclinical ketosis, many related to their etiology studies. However, borrowing from the classification of diabetes, according to blood ketone, glucose and free fatty acid level of ketosis was divided into type I. Disease and type of ketosis. At present, the metabolism of type II Group on dairy cow ketosis studies are rarely reported. Therefore, the research and application of NMR technology combined with multivariate statistical analysis, cow plasma on type I and type II ketosis ketosis change panoramic metabolism The aim of this study is to screen differentially Characteristic Metabolites of type I ketosis and type 2 ketosis in dairy cows, and to provide a scientific basis for enriching the etiology of cow type II ketosis and new strategies for prevention and treatment of dairy ketosis.
According to the study of plasma beta hydroxybutyric acid (BHBA), glucose (Glc) and free fatty acid (NEFA) level, select the 50 head test 7-28 days postpartum cows, type I ketosis group with no other diseases (K1, n=20 head) in the plasma of BHBA1.20mmol/L, Glc2.50mmol/L, NEFA0.50mmol/L; type II. The cow disease group (K2, n=20 head) in the plasma of BHBA1.20mmol/L, Glc2.80mmol/L, NEFA0.50mmol/L; the healthy control group (C, n=10) cow plasma BHBA1.00mmol/L, Glc3.75mmol/L, NEFA0.40mmol/L. NMR (1H Nuclear Magnetic Resonance, 1H NMR) plasma samples of three groups of test cows and metabolite detection, principal component analysis (Principle ComponentAnalysis, PCA), partial least squares method (Partial Least Squares-discriminant Analysis, PLS-DA) and orthogonal partial least squares method (Orthogonal Partial Least-Squares Discr IminantAnalysis, OPLS-DA) and multivariate statistical analysis, and application of biological analysis of specific metabolites and information technology biology. Finally using enzyme-linked immunosorbent assay (EnzymeLinked Immunosorbent, Assays, ELISA) of type I endogenous metabolites of ketosis and type II ketosis screened characteristic difference is verified.
Type I ketosis, type II ketosis and healthy control group there were significant differences between the three groups of cows. Metabolites identified 26 different metabolites, compared with healthy control, 2 kinds of elevated plasma substance disease type ketone for beta hydroxybutyric acid, acetone; 15 metabolites reduced to alanine, glutamic acid. Glutamine, glycine, histidine, lysine, citric acid, formic acid, lipid, creatine, myo inositol, O- acetyl glucosamine, choline phosphate, alpha beta glucose, glucose, there was a significant difference (P0.05). Compared with healthy controls, the plasma ketone disease type 3 metabolites or high beta hydroxybutyrate, acetone, lactic acid; 4 metabolites reduced to alanine, lysine, tyrosine, creatine, there are significant differences (P0.05). Compared with the type II ketosis, 8 elevated plasma metabolites disease type ketone for beta hydroxybutyric acid, acetic acid, acetone, isoleucine, leucine, valine, low density fat egg White and very low density lipoprotein; 14 metabolites reduced to alanine, citric acid, formic acid, glutamic acid, glutamine, glycine, histidine, lysine, inositol lipid, O-, N-acetylglucosamine, phosphocholine, alpha beta glucose, glucose, there is a significant difference (P0.05) between. Of Kyoto Encyclopedia of Genes andGenomes by metabolite (KEGG) database analysis, the detected metabolites involved in glucose metabolism, lipid metabolism and amino acid metabolism and energy metabolism, is also involved in the formation of ketone bodies. These differences in the metabolites isolated from cows with type I and type II ketosis metabolism are closely related to 11 different metabolites for pyruvate, alpha ketoglutarate, choline phosphoglyceride, oxaloacetic acid, citric acid, tyrosine, glycine, isoleucine, leucine, alanine, lactic acid, and the 11 metabolites were verified by ELISA method, the concentration of The result of the change coincide with the test results of 1H NMR technology.
The application of metabonomics technology 1H NMR screening and confirmation of the difference in plasma of type I and type II ketosis ketosis metabolites, not only verified the occurrence process of ketosis, metabolic disorder of lipid and amino acids, also set a theoretical foundation for the further study of the pathogenesis of ketosis in mechanism of prevention and treatment in future.

【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

【参考文献】