斑马鱼胚胎发育关键时期的代谢组变化规律的研究

Embryogenesis is a developmental process that usually begins once the egg has been fertilized. It involves multiplication of cells and their subsequent growth, movement, and differentiation into tissues and organs of a living. The process of embryonic development is a complex molecular regulatory network controlled by a series regulation of gene and protein. Previous research focused attentions on the changes of phenotypes and gene expression during the embryonic development. The importance of small endogenous metabolites was largely overlooked. The endogenous metabolites are the final products of gene regulation, and in the mean time, the alterations of the endogenous metabolites can in turn regulate gene expression. The abnormal changes of the small metabolites during embryonic development are closely associated with ill formation of organs. Therefore we propose the current project aiming to study the changes of the endogenous metabolites during the key stages of embryonic development using metabonomics technique..Metabonomics is a newly emerging analytical technique, which combines multivariate data analysis method with metabolic profiles generated from nuclear magnetic resonance (NMR) or mass spectrometry (MS). Since the birth of the technique, it has been applied to many research fields, such as the study of disease processes, drug toxicity, and interactions between animal hosts and parasites. The key tasks for metabonomics technique are to uncover metabonome information both quantitively and qualitively. Currently, profiling lipid metabolites is difficult for NMR technique due to heavy overlapping of lipid signals. Hence in the proposed project, we will first employ GC-MS method to develop a method that enables profiling of lipid. We will subsequently integrate NMR and MS metabolic profiling techniques to maximize metabolites detection and achieve integration between global metabolic profiling and targeted analysis. We will then employ zebrafish as a model and apply the newly developed technique to investigate alteration of metabonome during key stages of zebrafish embryonic development. By the combination of NMR and GC-MS metabolic profiles and multivariate data analysis methods, we will characterize the metabonome and monitor the formation and variation of the key endogenous metabolites throughout zebrafish embryonic development. Key metabolic pathways during the development will be elucidated. The methodology we demonstrated here could be applicable to other developmental studies and the successful conducting the proposed project will provide fundamental understanding of the molecular mechanism of zebrafish embryonic development. This project belongs to NSFC key funding areas of interdisciplinary, analysis and application of "-mics" in analytical chemistry.

胚胎发育是在一系列基因和蛋白的精密调控下，通过复杂的分子调控网络驱动进行的。内源性小分子代谢物作为基因调控过程的最终产物，它们的质和量的变化是生物整体在发育过程中对基因或环境的最终响应，同时小分子代谢物在体内的代谢和积累，又会反过来调控基因的表达，它们的异常变化与疾病发生密切相关。而目前对于胚胎发育过程中的代谢组的研究还很缺乏。本项目将运用代谢组学方法，以模式动物斑马鱼为对象，研究胚胎发育关键时期代谢组的变化规律。围绕这一关键科学问题，本项目将在发展NMR系统检测和GC-MS目标检测相结合的代谢组学方法基础上，获取斑马鱼胚胎发育关键时期的代谢指纹图谱，并结合多变量数据分析方法来研究胚胎发育关键时期的代谢谱特征、关键小分子代谢物形成过程及变化规律。本项目的顺利完成将为深入认识胚胎发育过程中的分子调控机制提供更多有价值的基础数据。本项目是属于分析化学中学科交叉、组学分析和应用的重点资助领域。

本研究项目主要通过基于NMR为基础的代谢组学和多变量统计分析相结合的方法，分析了斑马鱼胚胎发育过程中不同发育时期的代谢组变化及鱼类在受到温度及密度胁迫时的代谢应答规律。主要结果有：1.斑马鱼胚胎自受精（合子期）至孵化前，呈现出明显的发育规律。随着发育的不断进行，作为能源来源的糖元和葡萄糖等呈明显的下降趋势，且糖元与葡萄糖的比率在发育的中后期也呈现明显的下降；同时作为能量的载体物质-核苷酸类（ACP，ATP和肌苷）也出现明显的下降；但几乎所有的氨基酸类代谢物呈现明显的上升。表明斑马鱼主要是利用糖元和葡萄糖为发育的进行提供能量物质，氨基酸类物质是作为蛋白质合成的前体物质进行逐渐储备的，胚胎发育过程激活的关键通路主要涉及能量代谢过程。2.鱼类受到高温胁迫后其血液和肝脏代谢组发生了明显的改变。主要表现在血液中肌苷水平的显著下降和脂类的升高；甘氨酸、二甲基甘氨酸和高密度脂蛋白在高温处理的中后期呈现明显的下降，GPC、尿苷、缬氨酸、谷氨酰胺、葡萄糖、琥珀酸等也都发生了不同程度的变化。高温处理导致鱼类肝脏的牛磺酸水平显著降低，牛磺酸在动物的代谢中起着重要的生理作用，牛磺酸水平的降低也提示高温胁迫可能对鱼类的免疫系统造成负面影响。另外，在高温处理后，鱼类肝脏中Hsp70表达量显著上调，但上调的幅度随着胁迫作用时间的延长而下降。3.鱼类在不同养殖密度下也会有不同的代谢表现，与低密度组相比，高密度组的鱼类氨基酸代谢和能量代谢发生显著变化，说明密度作用下可能产生的胁迫导致了鱼类代谢组的改变。该项目较全面的分析了斑马鱼胚胎发育关键时期的代谢组变化特征及温度和密度胁迫对鱼类代谢生理的影响，表明基于NMR的代谢组学策略在鱼类胚胎发育等相关研究中能够提供更加丰富的信息。