色谱

色谱 ›› 2021, Vol. 39 ›› Issue (11): 1191-1202.DOI: 10.3724/SP.J.1123.2021.04009

• 研究论文 • 上一篇    下一篇

血清和血清外泌体的蛋白质组分析及其在肝内胆管癌中的应用

杨凯歌, 王薇薇, 王彦*(), 阎超*()   

  1. 上海交通大学药学院, 上海 200240
  • 收稿日期:2021-04-08 出版日期:2021-11-08 发布日期:2021-10-22
  • 通讯作者: 王彦,阎超
  • 作者简介:Tel:(021)38953588,E-mail: chaoyan@sjtu.edu.cn(阎超).
    * Tel:(021)34205673,E-mail: wangyan11@sjtu.edu.cn(王彦);
  • 基金资助:
    国家自然科学基金项目(81874307);国家自然科学基金项目(21874088);上海市科委“科技创新行动计划”(18142200700);上海市科委“科技创新行动计划”(19142203100);上海市科委“科技创新行动计划”(20142200400);细胞工程及抗体药物教育部工程研究中心开放课题(19X110020009-005);上海交通大学“新进青年教师启动计划”

Proteomic analysis of serum and serum exosomes, and their application in intrahepatic cholangiocarcinoma

YANG Kaige, WANG Weiwei, WANG Yan*(), YAN Chao*()   

  1. School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China
  • Received:2021-04-08 Online:2021-11-08 Published:2021-10-22
  • Contact: WANG Yan,YAN Chao
  • Supported by:
    National Natural Science Foundation of China(81874307);National Natural Science Foundation of China(21874088);Shanghai Science and Technology Commission “Science and Technology Innovation Action Plan”(18142200700);Shanghai Science and Technology Commission “Science and Technology Innovation Action Plan”(19142203100);Shanghai Science and Technology Commission “Science and Technology Innovation Action Plan”(20142200400);Open Project Program of Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, Shanghai Jiao Tong University(19X110020009-005);Startup Fund for Youngman Research at Shanghai Jiao Tong University

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摘要：

外泌体是由各种类型细胞在正常或非正常生理情况下分泌释放至细胞外且携带多种生物活性分子的细胞外囊泡,在细胞间通讯和免疫应答等生物过程中发挥着重要作用。肝内胆管癌是一种胆道上皮恶性肿瘤,早期无明显临床症状且生存率较低,目前常用的诊断手段包括依赖于影像设备的诊断方式和灵敏度及特异性较低的诊断标志物等,这些手段的不足对发展新的特异性标志物提出了需求。该文对血清中的外泌体进行了分离和表征,并采用液相色谱-质谱技术针对健康组与肝内胆管癌患者组的血清样本和血清外泌体样本进行了无标记定量蛋白质组学分析,分别从两种类型样本中鉴定并筛选到271和430种可信蛋白质。基于血清样本和血清外泌体样本的可信蛋白质定量表达值进行多维统计分析都能将健康组与肝内胆管癌患者组良好地区分开。对血清样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有15个上调和8个下调蛋白质;对血清外泌体样本中鉴定到的蛋白质进行差异蛋白质筛选,肝内胆管癌患者组相对于健康组有33个上调和18个下调蛋白质;基于两种样本筛选到的差异蛋白质中仅有4个是重复的,且基于血清外泌体样本的51个差异蛋白质中有35个蛋白质属于外泌体蛋白质数据库。针对差异蛋白质进行生物学信息分析,与差异蛋白质相关的分子功能、生物过程和信号通路主要涉及天然免疫反应、炎症反应和凝血等过程。该研究为发现肝内胆管癌的潜在生物标志物和探究肝内胆管癌的发生、发展和转移等过程提供了参考和借鉴价值。此外,通过比较研究发现血清外泌体样本能够获得较多的差异蛋白质和生物学信息,证明了外泌体作为组学分析样本的价值和应用潜力。

关键词: 液相色谱-质谱, 肝内胆管癌, 蛋白质组学, 统计模型, 差异蛋白质, 外泌体, 血清

Abstract:

Exosomes are extracellular vesicles with a diameter in the range of 50-200 nm and a double-layer lipid membrane structure that are released by various types of cells under normal or abnormal physiological conditions. At present, according to their extensive biological functions, exosomes have been used in a wide range of research fields and applications, including as potential biomarkers and drug delivery vehicles. Intrahepatic cholangiocarcinoma is a malignant tumor of the biliary epithelium with the characteristics of cholangiocellular differentiation, which accounts for 10%-15% of all types of primary liver cancer. Intrahepatic cholangiocarcinoma has no obvious clinical symptoms in the early stages, which results in a low survival rate. Imaging equipment dependent diagnostic methods and currently commonly used diagnostic markers with low sensitivity/specificity have necessitated the development of new specific markers for intrahepatic cholangiocarcinoma.
In this study, exosomes were isolated from serum using a commercial kit and characterized through nanoparticle tracking analysis, Western blotting analysis, and transmission electron microscopic analysis to prove the successful isolation of exosomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the protein profiles of the serum and serum exosome samples were significantly different. In particular, some high-abundance proteins in the serum samples were significantly reduced or disappeared in the serum exosome sample. Meanwhile, some protein bands (which may belong to exosomes) that did not appear in the serum samples appeared in the serum exosome samples, leading to the conduciveness of subsequent mass spectrometry analysis. The serum and serum exosome samples of the healthy control and intrahepatic cholangiocarcinoma groups were analyzed by liquid chromatography-mass spectrometry for label-free quantitative proteomics. In total, 547 proteins were identified in the serum exosome samples, of which 341 (more than 60%) could be found in the exosomal protein database. In addition, 271 and 430 credible proteins were screened from the serum and serum exosome samples for multi-dimensional statistical analysis and differential protein discovery. Unsupervised principal component analysis and supervised orthogonal partial least squares discriminant analysis based on the quantitative proteome of the serum and serum exosome samples could distinguish the healthy control and intrahepatic cholangiocarcinoma groups well, which illustrates that the two types of samples both have potential in the diagnosis of intrahepatic cholangiocarcinoma. There were 15 upregulated and 8 downregulated proteins screened in the intrahepatic cholangiocarcinoma group compared to the healthy control group based on the serum samples, while 33 upregulated and 18 downregulated proteins were screened in the intrahepatic cholangiocarcinoma group compared to the healthy control group based on the serum exosome samples, and only four of the differential proteins screened based on the two types of samples were duplicates. At the same time, 35 of the 51 differentially expressed proteins screened based on serum exosome samples belonged to the exosomal protein database. Finally, biological information analysis was performed according to these differential proteins. The molecular functions, biological processes, and signal pathways enriched by these differential proteins mainly involved the innate immune responses, inflammatory responses, and blood coagulation. This study provides a reference value for potential biomarker discovery and exploration of the process of occurrence, development, and metastasis of intrahepatic cholangiocarcinoma. Moreover, compared with proteomic analysis based on serum samples, proteomic analysis based on serum exosome samples can be used to identify more differential proteins and biological information, and although these differential proteins and biological information may show big differences, the specificity and sensitivity of exosome-based diagnosis and the superiority of exosomes as samples for proteomic analysis has proven the application value of exosomes.

Key words: liquid chromatography-mass spectrometry (LC-MS), intrahepatic cholangiocarcinoma, proteomics, statistical model, differential protein, exosome, serum

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