血清细胞外囊泡包含原发性硬化性胆管炎和胆管癌的蛋白质生物标志物。

Serum extracellular vesicles contain protein biomarkers for primary sclerosing cholangitis and cholangiocarcinoma.

机构信息

Department of Liver and Gastrointestinal Diseases, Biodonostia Research Institute, Donostia University Hospital, University of the Basque Country (UPV/EHU), San Sebastian, Spain.

Proteomics Platform, CIC bioGUNE, CIBERehd, ProteoRed-ISCIII, Bizkaia Science and Technology Park, Derio, Spain.

出版信息

Hepatology. 2017 Oct;66(4):1125-1143. doi: 10.1002/hep.29291. Epub 2017 Aug 26.

DOI:10.1002/hep.29291

PMID:28555885

Abstract

UNLABELLED

Cholangiocarcinoma (CCA) includes a heterogeneous group of biliary cancers with poor prognosis. Several conditions, such as primary sclerosing cholangitis (PSC), are risk factors. Noninvasive differential diagnosis between intrahepatic CCA and hepatocellular carcinoma (HCC) is sometimes difficult. Accurate noninvasive biomarkers for PSC, CCA, and HCC are not available. In the search for novel biomarkers, serum extracellular vesicles (EV) were isolated from CCA (n = 43), PSC (n = 30), or HCC (n = 29) patients and healthy individuals (control, n = 32); and their protein content was characterized. By using nanoparticle tracking analysis, serum EV concentration was found to be higher in HCC than in all the other groups. Round morphology (by transmission electron microscopy), size (∼180 nm diameter by nanoparticle tracking analysis), and markers (clusters of differentiation 9, 63, and 81 by immunoblot) indicated that most serum EV were exosomes. Proteome profiles (by mass spectrometry) revealed multiple differentially expressed proteins among groups. Several of these proteins showed high diagnostic values with maximum area under the receiver operating characteristic curve of 0.878 for CCA versus control, 0.905 for CCA stage I-II versus control, 0.789 for PSC versus control, 0.806 for noncirhottic PSC versus control, 0.796 for CCA versus PSC, 0.956 for CCA stage I-II versus PSC, 0.904 for HCC versus control, and 0.894 for intrahepatic CCA versus HCC. Proteomic analysis of EV derived from CCA human cells in vitro revealed higher abundance of oncogenic proteins compared to EV released by normal human cholangiocytes. Orthotopic implant of CCA human cells in the liver of immunodeficient mice resulted in the release to serum of EV containing some similar human oncogenic proteins.

CONCLUSION

Proteomic signatures found in serum EV of CCA, PSC, and HCC patients show potential usefulness as diagnostic tools. (Hepatology 2017;66:1125-1143).

摘要

目的

胆管癌（CCA）包括一组预后不良的异质性胆道癌，一些情况如原发性硬化性胆管炎（PSC）是其危险因素。肝内 CCA 与肝细胞癌（HCC）的非侵入性鉴别诊断有时较为困难，尚缺乏用于 PSC、CCA 和 HCC 的准确非侵入性生物标志物。在寻找新的生物标志物时，我们从 CCA（n=43）、PSC（n=30）或 HCC（n=29）患者和健康个体（对照组，n=32）中分离了血清细胞外囊泡（EV），并对其蛋白含量进行了特征分析。通过纳米颗粒跟踪分析，发现 HCC 患者的血清 EV 浓度高于其他所有组。通过透射电子显微镜观察到的圆形形态、（纳米颗粒跟踪分析测定的）~180nm 直径大小以及（免疫印迹分析的）标志物（CD9、CD63 和 CD81 簇）表明大多数血清 EV 是外泌体。通过质谱分析（mass spectrometry）揭示了各组之间存在多个差异表达蛋白。其中一些蛋白的诊断价值较高，CCA 与对照组比较的曲线下面积最大为 0.878，CCA Ⅰ-Ⅱ期与对照组比较的曲线下面积最大为 0.905，PSC 与对照组比较的曲线下面积最大为 0.789，非肝硬化性 PSC 与对照组比较的曲线下面积最大为 0.806，CCA 与 PSC 比较的曲线下面积最大为 0.796，CCA Ⅰ-Ⅱ期与 PSC 比较的曲线下面积最大为 0.956，HCC 与对照组比较的曲线下面积最大为 0.904，肝内 CCA 与 HCC 比较的曲线下面积最大为 0.894。对体外培养的 CCA 人细胞衍生的 EV 进行蛋白质组学分析发现，与正常人类胆管细胞释放的 EV 相比，其含有更高丰度的致癌蛋白。在免疫缺陷小鼠肝脏原位植入 CCA 人细胞导致含有一些相似的人类致癌蛋白的 EV 释放到血清中。