线粒体代谢组分析新方法的建立及在肝癌细胞研究中的应用

Mitochondria are the main sites of cell metabolism, which provides energy for cells and participates in a variety of physiological and pathological activities. Systematic study of mitochondrial metabolic profiling is favorable to understanding mitochondrial metabolism and its important role in disease. The important matter in mitochondrial metabolomics study is to establish a rapid and reliable isolation method and develop analytical approach with high sensitivity and broad coverage for profiling acquisition. This project intends to utilize interactions between magnetic nanoparticles and His-6 tagged mitochondrial outer membrane protein (OMP25) to establish a rapid and stable isolation method for mitochondria. And isotopic tracing of tricarboxylic acid cycle pathway is applied to evaluate the change of mitochondrial metabolism during isolation process. Further, in order to improve sensitivity and coverage of mitochondrial metabolites detection, CE-MS based method for complete analysis of mitochondrial polar metabolites and LC-MS based method combined with chemical derivatization for metabolites containing hydroxyl, carboxyl and amino groups are established for mitochondrial metabolomics study. The developed method is applied to mitochondrial metabolome analysis of liver cancer cells. Metabolic profiling of hepatocellular carcinoma cells and their mitochondria are acquired to obtain metabolic panorama and clarify their metabolic differences. The implementation of this project will not only provide a new method for metabolomics study of mitochondria, but also reveal metabolic characteristics of liver cancer at subcellular level.

线粒体是细胞代谢的主要场所，为细胞提供能量，并参与多种生理病理活动。线粒体代谢谱的系统研究有助于深入了解线粒体代谢及其在疾病中发挥的作用。线粒体代谢组学研究的关键问题是建立快速、可靠的线粒体分离提纯方法及高灵敏、高覆盖的组学分析方法。本项目拟利用磁性纳米材料表面键合的Ni2+与线粒体外膜蛋白（OMP25）表位标记的His-6蛋白标签间的亲和作用建立线粒体分离提纯方法，并通过同位素示踪三羧酸循环通路评价线粒体提取过程中自身代谢的变化程度。建立基于CE-MS的极性代谢组的全组分分析方法和基于LC-MS结合化学衍生的含羟基、羧基及氨基代谢物的分析方法，以提高线粒体代谢物的检测灵敏度和覆盖度。采用所建立的方法对肝癌细胞线粒体进行分离分析，获取整细胞和线粒体的代谢概览图，阐明整细胞与亚细胞的代谢差异。本项目的实施能够为线粒体代谢组学研究提供新方法，并揭示肝癌亚细胞水平的代谢特征。

线粒体是细胞代谢的主要场所，为细胞提供能量，并参与多种生理病理活动。线粒体代谢谱的系统研究有助于深入了解线粒体代谢及其在疾病中发挥的作用。首先开发了基于羧甲基化的聚乙烯亚胺修饰的磁性纳米材料的线粒体分离提取方法。鉴于磁性纳米材料具有比表面积大、表面易修饰、操作简便等优势，首先合成Fe3O4磁性纳米材料核，然后在磁性材料表面键合上羧甲基化聚乙烯亚胺（CMPEI），以提供大量反应位点结合Ni2+，实现高效的富集带有His标签的蛋白，可用于提取转染了His标签的细胞中的线粒体。还开发了一种抗体修饰的磁性亲和材料并将其用于肝细胞中线粒体的分离与纯化。该材料集成了四氧化三铁的外磁场可控、石墨烯的大表面积、聚多巴胺对NH2的反应活性、抗体对线粒体外膜蛋白的高特异性识别等多种优势，可有效分离细胞中的线粒体。在分析方法方面，建立了非靶向修饰代谢物的高覆盖检测方法，该方法整合多种数据采集模式，有效地扩大了修饰代谢物的检出覆盖度，可同时检测13大类修饰类型。还开发了基于化学衍生化的甾体激素的检测方法，实现了低丰度重要代谢物的高灵敏检测。以肝癌细胞为研究对象，采用建立的线粒体分离提取方法分离细胞中的线粒体，并进行代谢组学分析，在亚细胞水平阐明肝癌细胞的代谢特征。本项目的实施为亚细胞水平的代谢组学研究提供了新方法。