束缚应激致大鼠高同型半胱氨酸血症的肝脏代谢组学研究

发布时间：2018-03-02 00:28

本文关键词： 束缚应激高同型半胱氨酸血症代谢组学肝脏核磁共振主成分分析法　出处：《华北煤炭医学院》2009年硕士论文　论文类型：学位论文

【摘要】：目的通过束缚应激的建模方法建立大鼠高同型半胱氨酸血症的模型,探讨大鼠高同型半胱氨酸血症肝脏代谢模式的变化;揭示机体中同型半胱氨酸含量显著升高引起肝脏代谢紊乱的相关作用机制;寻求代谢标志物以及作用靶点。方法选用健康雄性Wistar大鼠,体重180~200g,随机分为对照组和应激组。将应激组大鼠分为5个应激强度不同的时间组,分别束缚1周、2周、4周、6周和8周,建立应激动物模型。检测血浆中的糖皮质激素、儿茶酚胺和同型半胱氨酸的含量。采集模型动物的肝脏组织,采用萃取法分别提取肝脏组织中的水溶性提取物和脂溶性提取物,利用1~H核磁共振技术对样品进行检测并通过主成分分析法(PCA)比较正常对照组和应激组的大鼠肝脏组织中水溶性提取物和脂溶性提取物的代谢模式,建立稳定的肝脏组织代谢组学的技术平台。通过对模型动物肝脏组织的代谢组学研究,探讨鼠龄和应激强度对肝脏代谢状态的影响和代谢模式的差异。根据因子载荷图(loading plot)和文献及数据库检索鉴定出影响代谢模式变化的贡献量大的代谢小分子。并对这些代谢小分子的代谢途径进行分析,探讨代谢小分子在机体发生肝脏代谢紊乱时的作用机制以及与高同型半胱氨酸血症的相关性。结果通过束缚应激的方法建立了高同型半胱氨酸血症的动物模型。建立了高重复性和高分辨率的大鼠肝脏组织提取液的代谢组学的研究方法。通过比较正常和应激后肝脏组织1~H-NMR谱图和PCA分析,发现不同应激强度的大鼠肝脏代谢模式明显不同并与同鼠龄的对照组大鼠的肝脏代谢模式差异显著,代谢成分也可明显区分开。大鼠肝脏组织水溶性提取物中有24种代谢小分子的含量发生变化,如乳糖、β-葡萄糖、琥珀酸等;脂溶性提取物中14种代谢小分子的含量发生变化,如LipidCH3、Lipid (CH2 )n、LipidCH2CH2CO等。这些代谢成分的改变提示束缚应激可导致能量代谢中糖酵解、糖异生能力、三羧酸循环的改变以及肝脏脂代谢的紊乱。其肝脏代谢紊乱可能与与线粒体、内质网功能降低、氧化磷酸化、脂肪氧化改变以及肝脏抗氧化防御体系受损有关。结论束缚应激导致大鼠高同型半胱氨酸血症的发生;经过1~H-NMR的检测和PCA分析后发现束缚应激致高同型半胱氨酸血症大鼠肝脏的代谢状态发生了改变,随着应激强度的增加大鼠肝脏的代谢表型也发生显著性变化;经鉴定后发现的对代谢表型改变贡献量大的代谢小分子(氨基酸、脂溶性化学结构片段,如谷氨酸、丙氨酸、LipidCH2C=C、LipidCH2CO)可对束缚应激导致的高同型半胱氨酸血症机体的能量代谢产生巨大影响。
[Abstract]:Objective to establish a rat model of hyperhomocysteinemia by restraint stress and to explore the changes of hepatic metabolic pattern of hyperhomocysteinemia in rats. To reveal the related mechanism of hepatic metabolic disorder caused by the increase of homocysteine content in the body, and to seek for metabolic markers and action targets. Methods healthy male Wistar rats, weighing 180g, were randomly divided into control group and stress group. The rats in the stress group were divided into 5 groups with different stress intensity, one week, two weeks, four weeks, six weeks and eight weeks, respectively. Stress animal model was established. The levels of glucocorticoid, catecholamine and homocysteine in plasma were determined. The liver tissue of the model animal was collected and the water-soluble and fat-soluble extracts from the liver tissue were extracted by extraction method. The metabolic patterns of water-soluble extracts and liposoluble extracts in the liver tissues of rats in normal control group and stress group were compared by using 1H NMR technique and PCAA (principal component analysis). To establish a stable technical platform of liver tissue metabolomics. By studying the metabolomics of liver tissue in model animal, To explore the effect of age and stress intensity on the metabolic state of liver and the difference of metabolic model, small metabolic molecules with large contribution to the change of metabolic pattern were identified according to the factor load map loading plot), literature and database. To analyze the metabolic pathways of these metabolic small molecules, To explore the mechanism of metabolic small molecules in the occurrence of liver metabolic disorder and the correlation with hyperhomocysteinemia. Results the animal model of hyperhomocysteinemia was established by restraint stress. A high reproducibility and high resolution method of metabolomics of rat liver tissue extract was established. 1H-NMR and PCA analysis of liver tissue after stress. It was found that the liver metabolic patterns of rats with different stress intensities were significantly different from those of control rats of the same age. The contents of 24 kinds of metabolic small molecules, such as lactose, 尾 -glucose, succinic acid and so on, were changed in the water-soluble extracts of rat liver, and the contents of 14 kinds of metabolic small molecules in liposoluble extracts were changed. For example, LipidCH3, Lipid CH2, LipidCH2CH2CO and so on. The changes of these metabolic components suggest that restraint stress may lead to glycolysis, glycosylation, changes in tricarboxylic acid cycle and disorder of hepatic lipid metabolism in energy metabolism. Decreased endoplasmic reticulum function, oxidative phosphorylation, lipid oxidation and damage to the liver antioxidant defense system. Conclusion restraint stress leads to the occurrence of hyperhomocysteinemia in rats, and after 1H-NMR detection and PCA analysis, it is found that the metabolic state of liver of rats with hyperhomocysteinemia induced by restraint stress has changed. With the increase of stress intensity, the metabolic phenotype of rat liver also changed significantly, and the small metabolic molecules (amino acids, liposoluble chemical structure fragments, such as glutamic acid, glutamic acid, etc.) that contributed to the change of metabolism phenotype were found after identification. LipidCH2C) has a great effect on the energy metabolism of hyperhomocysteinemia induced by restraint stress.
【学位授予单位】：华北煤炭医学院
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R363

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