多分析方法与多样本整合的代谢组学研究食管癌淋巴结转移生物标志物

Lymph node metastasis was an important prognostic factor for esophageal cancer. At present, the mechanism of lymph node metastasis in esophageal cancer is still not clear. The discovery of biomarkers for lymph node metastatic esophageal cancer is important for clinical diagnosis and treatment. In this project, we will use the integrated metabolomics strategy including different analytical methods and samples to look for sensitive and specific diagnostic biomarkers for esophageal cancer with lymph node metastasis. First, a ultra performance liquid chromatography combined with quadruple/time-of-flight mass spectrometry (UPLC-QTOFMS) method coupled with a proton nuclear magnetic resonance (1H NMR) spectroscopy method was developed to characterize the metabolic features of esophageal cancer. Metabolomic profiling of pairs of tissue samples and matched normal tissues including distal noncancerous tissues, tumor tissues and lymph node tumor tissues collected from lymph node metastatic esophageal cancer patients were performed by using both UPLC-QTOFMS and 1H NMR, and differential metabolites were filtered and identified from the tissue metabolome. Then，differential metabolites identified in tissues were tested in serum specimens from lymph node metastatic esophageal cancer patients, esophageal cancer patients and the healthy controls using a ultra high pressure liquid chromatography combined with tandem mass spectrometry (UPLC-MS/MS) based targeted metabolomics method to confer their good diagnostic potential. This project is expected to help us to understand the metabolic mechanism of lymph node metastasis, and provide a theoretical basis for esophageal cancer clinical diagnosis, accurate staging, treatment and prognosis.

淋巴结转移是影响食管癌预后的重要因素。目前食管癌发生淋巴结转移的机制仍不明确。发现食管癌淋巴结转移相关生物标志物对辅助临床诊断和治疗具有重要意义。本项目拟采用多分析检测方法与多样本整合的代谢组学研究思路：1）建立基于UPLC-QTOFMS和1H NMR技术的食管癌组织非靶向代谢组学分析方法。2）对食管癌淋巴结转移患者远端癌旁组织、癌组织及转移淋巴结进行非靶向代谢组学分析，筛选对淋巴结转移及预后有预警意义的差异代谢物，研究相关代谢通路。3）针对组织样本中的差异代谢物，建立基于UPLC-MS/MS技术的血清靶向代谢组学分析方法。4）对正常人、食管癌无淋巴结转移患者及食管癌淋巴结转移患者的血清进行靶向代谢组学分析，验证差异代谢物在血清中的诊断能力，进一步筛选出食管癌淋巴结转移特异性生物标志物。本研究有助于深入理解食管癌淋巴结转移的代谢机制，为食管癌临床诊断、病理分期、治疗和预后评估提供科学依据。

本项目建立了基于高分辨 1H 核磁共振（1H NMR）技术和超高效液相色谱-三级四极杆飞行时间质谱联用（UPLC-QTOF/MS）技术的非靶向代谢组学分析方法，研究了食管癌患者的血清和组织代谢组学特点，项目还将建立的方法用于非小细胞肺癌的代谢组学研究。研究发现，采用基于1H NMR技术的代谢组学分析能清晰地区分食管癌患者癌组织和远端癌旁组织，癌组织中糖酵解代谢显著增加。另外，氨基酸代谢、核苷代谢、胆碱代谢、三羧酸（TCA）循环、能量代谢等通路均发生不同程度的紊乱。采用UPLC-QTOF/MS非靶向代谢组学分析方法，初步考察了食管癌患者和正常健康人血清的代谢组学差异。研究发现，食管癌患者体内嘌呤代谢、色氨酸代谢、氨基酸代谢、饱和及不饱和脂肪酸代谢、花生四烯酸代谢、细胞色素代谢等均发生了不同程度紊乱。这些结果与前期食管癌患者血清代谢组学研究结果是相一致的，且得到了一些互补信息。由于本项目侧重于分析方法的建立和方法学应用，因此除食管癌患者的代谢组学研究之外，本项目还将建立的非靶向代谢组学研究方法应用于非小细胞肺癌患者的血清代谢组学研究。研究发现，肺癌患者与正常健康人血清存在明显的代谢差异，这些差异代谢物可能为肺癌的临床诊断、治疗提供依据。研究提示，本项目所建立的代谢组学研究方法可用于鉴别和发现与肿瘤诊断、治疗相关的代谢标记物。项目执行期间，使用项目建立的代谢组学分析方法，发表SCI论 文1篇；培养毕业硕士研究生2 人；项目组成员参加国内、国际相关学术会议9人次。