基于稳定同位素示踪和LC-MS的小鼠动态代谢分析新方法及其在肝癌研究中的应用

Dysregulated metabolism is an important characteristic of cancer. The dynamic metabolic analyses which incorporate the stable isotope tracing focus on the dynamic change of anabolism/catabolism of each metabolite in the specific pathway and the activities of related enzymes, which will be helpful for the interpretation of cancer mechanism. Currently, the tumor dynamic metabolic researches are conducted mainly on cell cultures, the few animal studies are focused on central carbon metabolism, which overlook the distinctly dysregulated tumor lipid metabolism. Previously we used 13C16-palmitate as the stable isotope tracer to study the dynamic lipid metabolism in cell culture, and through another research we found that hepatocellular carcinoma was associated with aberrant amino acid and lipid metabolism. In this project, we plan to develop a new method to analyze the dynamic metabolism in mouse, which incorporate the stable isotope tracing in vivo and liquid chromatography-high resolution mass spectrometry analysis. 13C6-glucose and 13C16-palmitate will be injected together to the mouse, then the tissue/plasma metabolites will be extracted with a methyl tert-butyl ether/methanol/water-based biphasic extraction system, and the dynamic central carbon metabolism and lipid metabolism will be analyzed by hydrophilic interaction liquid chromatography and reversed phase chromatography, respectively, both are hyphenated to high resolution mass spectrometry. The developed method will be applied to the orthotopic mouse model of hepatocellular carcinoma (HCC) to demonstrate its practicability in cancer research, in the meantime the dynamic metabolic changes and metabolic pathway modulation during HCC development will also be explored.

代谢异常是癌症的一个重要特征。结合稳定同位素示踪的动态代谢分析侧重于研究代谢途径上各代谢物的合成/分解动态变化及相关代谢酶的活性，有助于从机理上对癌症进行解释。当前肿瘤动态代谢研究主要是在细胞培养层面开展，仅有的动物模型研究也集中于中心碳代谢上，而对肿瘤中发生显著紊乱的脂质代谢则少有关注。我们前期通过13C16-棕榈酸示踪研究了细胞的动态脂质代谢，另外研究发现肝癌病人发生了氨基酸和脂质代谢异常。在本项目中我们计划通过结合稳定同位素示踪和LC-MS建立小鼠动态代谢分析的新方法：将13C6-葡萄糖和13C16-棕榈酸联合注射给小鼠，其组织/血浆代谢物经甲基叔丁基醚/甲醇/水双相体系提取后分别利用亲水作用色谱和反相液相色谱与高分辨质谱联用全面分析其动态中心碳代谢和脂质代谢。将建立的方法用于肝癌小鼠原位移植瘤模型来展示其在肿瘤研究中的实用性，并探索肝癌发生发展相关的动态代谢变化及其代谢途径的调变。

本项目致力于建立全面分析小鼠动态中心碳代谢和脂质代谢的方法，并将其应用于肝癌小鼠原位移植瘤模型，来探索肝癌发生发展相关的动态代谢变化及其代谢途径的调变。在项目实施过程中，我们通过腹腔注射13C6-葡萄糖建立了小鼠动态代谢模型，其肝脏组织/血浆代谢物经甲基叔丁基醚/甲醇/水双相体系提取后，通过气相色谱-质谱和超高效液相色谱-高分辨质谱分别分析其中心碳代谢物和脂质代谢物，构建了小鼠全面动态代谢分析的新方法。所建立的方法可用于研究肿瘤小鼠模型的全面动态代谢，探索肿瘤发病机制和治疗靶点。同时开发了一种经过一次提取，可以同时获得小量组织样本中丰富的代谢组、脂质组和蛋白质组信息的方法；所开发方法不仅节约了样本用量，也保证了组织样本多组学分析时的数据一致性。另外通过脂质组学研究发现胆囊癌脂质代谢发生显著紊乱，其中酰基肉碱代谢相关的肉碱-棕榈酰转移酶具有明显的促癌作用，并可能通过影响HIF1a通路发挥作用。