先天性畸形羊水代谢组学研究

发布时间：2018-02-27 06:24

  本文关键词： 先天性畸形 代谢组学 超高效液相色谱联合质谱 气相色谱串联质谱 模式识别　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：代谢组学(Metabolomics或Metabonomics)是近年来在系统生物学蓬勃开展的背景下发展起来的一门新兴学科。目前国内外将代谢组学分为4个层次：代谢物靶标分析、代谢轮廓(谱)分析、代谢组学和代谢指纹分析。其中，代谢指纹分析(metabolic finger printing)是以整个图谱(指纹图谱)代表特定细胞或组织的特定代谢模式,不分离鉴定具体单一组分,是一种快速、高通量、全局分析手段,因此样本制备相对简单。代谢指纹分析通常结合模式识别和判别技术用来分类代谢指纹和识别不同模式指纹的某些特征,是在不同状态判别筛选、疾病诊断及其特定代谢模式的发现中最有用的方法。代谢指纹分析能通过反映整体的代谢指纹图谱监测体内代谢网络的运行状态、诊断疾病、评价疗效、预测人体未来的健康发展趋势。因此，代谢组学研究应用于产前诊断具有十分重要意义，本文建立了两种方法（超高效液相色谱联合质谱以及气相色谱质谱）对先天性畸形羊水代谢进行详细研究，内容如下。 1.基于超高效液相色谱联合质谱对先天性畸形羊水代谢组学轮廓研究 先天性畸形是婴儿死亡主要原因之一，它可能与基因、环境等因素有关，但通过传统检查方法很难解释其发病机制。代谢组学研究生物系统变化，能够发现与疾病相关的小分子。因此本文建立基于超高效液相色谱联合质谱（UPLC-MS）联合多种识别模式对先天性畸形羊水和正常羊水代谢组学轮廓研究，鉴定出14种物质，它们都涉及重要的代谢途径，包括亮氨酸代谢、谷氨酸代谢、丝氨酸代谢、组氨酸、精氨酸、苯丙氨酸代谢以及能量代谢。这些发现可能对于研究先天性畸形发病机制有一定价值，，同时可以为产前诊断提供新的代谢标志物。 2.基于气相色谱串联质谱对先天性畸形羊水中氨基酸定量分析的研究 氨基酸在许多代谢通路中有重要作用，如核酸、维生素、蛋白质的生物合成。本文通过运用气相色谱联合质谱（GC-MS）及柱前三甲基硅烷衍生化检测先天性畸形羊水中的氨基酸，最终检测出8种氨基酸，其中亮氨酸、异亮氨酸、丝氨酸及苏氨酸差异性较大，可能与先天性畸形关系密切。同时对衍生化时间和温度进行优化，在最优条件下（70℃以及60min），氨基酸在0.5μg/ml-10μg/ml浓度范围内呈线性关系，相关系数大于0.99，该方法的精密度0.84%-9.33%，回收率91.12%-104.41%，该方法灵敏性、精密度较好。研究结果表明先天性畸形羊水中氨基酸改变可能是其致病发展的原因之一，该研究结果可以让我们多了解先天性畸形发病机制。
[Abstract]:Metabolomics or Metabonomicsis a new subject developed in the background of the vigorous development of systems biology in recent years. At present, the credit of metabolic group is divided into four levels at home and abroad: metabolite target analysis, metabolic profile (spectrum) analysis. Metabonomics and fingerprinting. Metabolic finger printings represent a specific metabolic pattern of a particular cell or tissue, and do not separate and identify a single component, which is a fast, high-throughput. Metabolic fingerprint analysis is usually combined with pattern recognition and discriminant technology to classify metabolic fingerprint and identify some characteristics of different pattern fingerprints. The most useful method in disease diagnosis and the discovery of specific metabolic patterns. Metabolic fingerprinting can monitor the operating status of metabolic networks in the body, diagnose diseases, and evaluate the efficacy by reflecting the overall metabolic fingerprint. Therefore, the application of metabonomics in prenatal diagnosis is of great significance. In this paper, two methods (ultra-high performance liquid chromatography combined mass spectrometry and gas chromatography-mass spectrometry) were established to study the amniotic fluid metabolism in congenital malformation. 1. To study the metabolic profile of amniotic fluid in congenital malformation based on ultra-high performance liquid chromatography and mass spectrometry. Congenital malformation is one of the main causes of infant death, it may be related to genes, environment and other factors, but it is difficult to explain its pathogenesis by traditional methods. Therefore, based on UPLC-MS) and multiple recognition patterns, the metabolic profiles of congenital malformed amniotic fluid and normal amniotic fluid were studied, and 14 substances were identified. They all involve important metabolic pathways, including leucine metabolism, glutamate metabolism, serine metabolism, histidine, arginine, These findings may be valuable in studying the pathogenesis of congenital malformation and may provide a new metabolic marker for prenatal diagnosis. 2. Quantitative analysis of amino acids in congenital amniotic fluid based on gas chromatography tandem mass spectrometry. Amino acids play an important role in many metabolic pathways, such as the biosynthesis of nucleic acids, vitamins and proteins. In this paper, amino acids in congenital malformed amniotic fluid were detected by gas chromatography-mass spectrometry (GC-MS) and pre-column trimethylsilane derivation. Finally, eight kinds of amino acids were detected, including leucine, isoleucine, serine and threonine, which may be closely related to congenital malformation. At the same time, the derivation time and temperature were optimized. Under the optimum conditions of 70 鈩

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