發(fā)布時(shí)間：2018-03-25 17:11

  本文選題：Ⅱ型酮病　切入點(diǎn)：Ⅰ型酮病　出處：《黑龍江八一農(nóng)墾大學(xué)》2015年碩士論文

【摘要】：酮病是圍產(chǎn)期高產(chǎn)奶牛常見而多發(fā)的一種能量代謝障礙性疾病。本病主要以低血糖、高酮體和高游離脂肪酸為生化特征；能量負(fù)平衡（Negative energy balancen, NEB）、脂肪過(guò)度動(dòng)員為本病的病理學(xué)基礎(chǔ)。通常根據(jù)血中酮體水平及臨床癥狀，奶牛酮病被分為臨床型酮病以及亞臨床型酮病，有關(guān)它們的病因?qū)W研究已有很多。然而，借鑒于糖尿病的分類，根據(jù)血中酮體、葡萄糖以及游離脂肪酸的水平，奶牛酮病又被分為Ⅰ型酮病和Ⅱ型酮病。目前，關(guān)于奶牛Ⅱ型酮病的代謝組學(xué)研究報(bào)道極少。因此，本研究應(yīng)用核磁共振技術(shù)并結(jié)合多元統(tǒng)計(jì)分析，對(duì)Ⅰ型酮病和Ⅱ型酮病的奶牛血漿進(jìn)行全景式代謝變化的研究，旨在篩選出奶牛Ⅰ型酮病和Ⅱ型酮病的差異性特征性代謝物，為豐富奶牛Ⅱ型酮病的病因?qū)W以及奶牛酮病防治新策略提供科學(xué)依據(jù)。 本研究根據(jù)血漿中β-羥丁酸（BHBA）、葡萄糖（Glc）和游離脂肪酸（NEFA）的水平，選取了50頭產(chǎn)后7-28天試驗(yàn)?zāi)膛�，其中無(wú)其他疾病的Ⅰ型酮病組奶牛（K1，n=20頭）血漿中BHBA1.20mmol/L，Glc2.50mmol/L，NEFA0.50mmol/L；Ⅱ型酮病組奶牛（K2，n=20頭）血漿中BHBA1.20mmol/L，Glc2.80mmol/L, NEFA0.50mmol/L；健康對(duì)照組奶牛（C，n=10頭）血漿中BHBA1.00mmol/L，Glc3.75mmol/L，NEFA0.40mmol/L。先運(yùn)用核磁共振技術(shù)（1H Nuclear Magnetic Resonance，1H NMR）對(duì)三組試驗(yàn)?zāi)膛５难獫{樣品進(jìn)行代謝物檢測(cè)，而后進(jìn)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）等多元統(tǒng)計(jì)分析，再應(yīng)用生物學(xué)信息技術(shù)對(duì)差異性代謝物進(jìn)行生物學(xué)分析。最后采用酶聯(lián)免疫（EnzymeLinked Immunosorbent Assays，ELISA）方法對(duì)Ⅰ型酮病和Ⅱ型酮病篩選出的內(nèi)源特征性差異代謝物進(jìn)行驗(yàn)證。 Ⅰ型酮病、Ⅱ型酮病與健康對(duì)照組奶牛三組間代謝成分存在顯著差異。鑒定出26種差異代謝物，相對(duì)于健康對(duì)照，Ⅰ型酮病血漿中升高的2種物質(zhì)為β-羥丁酸、丙酮；降低的15種代謝物為丙氨酸、谷氨酸、谷氨酰胺、甘氨酸、組氨酸、賴氨酸、檸檬酸、肌酸、甲酸、脂質(zhì)、肌醇、O-乙酰葡萄糖胺、磷酸膽堿、α-葡萄糖、β-葡萄糖，有顯著的差異(P0.05)。相對(duì)于健康對(duì)照，Ⅱ型酮病血漿中升高的3種代謝物為β-羥丁酸、丙酮、乳酸；降低的4種代謝物為丙氨酸、賴氨酸、酪氨酸、肌酸，有顯著的差異(P0.05)。與Ⅱ型酮病相比，Ⅰ型酮病血漿中升高的8種代謝物為β-羥丁酸、乙酸、丙酮、異亮氨酸、亮氨酸、纈氨酸、低密度脂蛋白和極低密度脂蛋白；降低的14種代謝物為丙氨酸、檸檬酸、甲酸、谷氨酸、谷氨酰胺、甘氨酸、組氨酸、脂質(zhì)、賴氨酸、肌醇、O-乙酰葡萄糖胺、磷酸膽堿、α-葡萄糖、β-葡萄糖，有顯著的差異(P0.05)。將差異性代謝物通過(guò)Kyoto Encyclopedia of Genes andGenomes（KEGG）數(shù)據(jù)庫(kù)分析，檢測(cè)到的代謝物不僅參與糖代謝、脂肪代謝和氨基酸代謝等能量代謝，也參與酮體的形成。從這些差異性代謝物中篩選出與奶牛Ⅰ型和Ⅱ型酮病代謝密切相關(guān)的11種差異性代謝物為丙酮酸、α-酮戊二酸、膽堿磷酸甘油酯、草酰乙酸、甘氨酸、酪氨酸、檸檬酸、異亮氨酸、亮氨酸、丙氨酸、乳酸，并用ELISA方法對(duì)這11種差異代謝物進(jìn)行驗(yàn)證，其濃度變化結(jié)果與1H NMR技術(shù)的檢測(cè)結(jié)果相符。 應(yīng)用代謝組學(xué)的1H NMR技術(shù)篩選和確證了Ⅰ型酮病和Ⅱ型酮病的血漿差異代謝物，不僅驗(yàn)證了奶牛酮病發(fā)生過(guò)程中糖、脂類和氨基酸的代謝紊亂，，也為今后進(jìn)一步探究Ⅱ型酮病的發(fā)病機(jī)制和防治奠定理論基礎(chǔ)。
[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.

【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.23

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