应用蛋白质组学技术筛选、鉴定循环肝癌干细胞特异性标志物

项目名称： 应用蛋白质组学技术筛选、鉴定循环肝癌干细胞特异性标志物

项目编号： No.30801342

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 金属学与金属工艺

项目作者： 徐文

作者单位： 中国人民解放军第二军医大学

项目金额： 20万元

中文摘要： 创建了一种独特的基于去唾液酸糖蛋白受体（asialoglycoprotein receptor，ASGPR）及其配体相互作用的肝癌循环肿瘤细胞（circulating tumor cells，CTCs）磁性分离方法。研究结果不仅表明肝癌CTCs检测具有评估疾病进程和预测肝癌转移复发、生存预后的潜能，并且发现肝癌CTCs是具有高度异质性的细胞群体，并存凋亡细胞和未凋亡细胞。部分CTCs 具有HER-2 基因扩增和TP53 基因缺失现象。从分离的CTCs 中可检出CD133 阳性细胞。 应用球培养法从多种肝癌细胞系富集肿瘤干细胞（cancer stem cell，CSCs）样球细胞。并进行一系列特征鉴定。结果显示，PLC/PRF/5 球细胞具有持续自我更新能力、无限增殖能力、耐药性、高表达肝癌CSCs相关蛋白（Oct3/4，OV6，EpCAM，CD133，CD44），最少500个球细胞即可在NOD/SCID小鼠皮下成瘤。CSL-非依赖性Notch信号通路的下游蛋白DTX1和Ep300在球细胞中高表达，而采用MRK003阻断Notch信号通路可以明显抑制球形成能力。

中文关键词： 肝癌；循环肿瘤细胞；肿瘤干细胞；生物标志物

英文摘要： We report the development and validation of a unique magnetic cell separation system, which allows enumeration, immunomorphologic identification, and genetic analysis of circulating tumor cells (CTCs) from peripheral blood samples of HCC patients, mediated by the interaction of the asialoglycoprotein receptor exclusively expressed on hepatocytes with its ligand. The system was used to detect CTCs in 5 mL blood. TP53 gene deletion and HER-2 amplification were observed in CTCs. CD133-positive CTCs were detected in some HCC patients. The data from this study show that this system has high sensitivity and specificity and is likely clinically useful in improving prognostic accuracy, monitoring therapeutic outcomes, and treatment decision making for HCC. The human hepatoma cell lines PLC/PRF/5, MHCC97H and HepG2 cells could form clonal nonadherent 3-D spheres in stem cell conditioned culture system and be serially passaged. The PLC/PRF/5 sphere-forming cells possessed a key criteria that define CSCs: persistent self-renewal, extensive proliferation, drug resistance, overexpression of liver CSCs related proteins (Oct3/4, OV6, EpCAM, CD133 and CD44). Even 500 sphere-forming cells were able to form tumors in NOD/SCID mice. Besides, downstream proteins DTX1 and Ep300 of the CSL-independent Notch signaling pathway were highly expressed in the spheres, and a gamma-secretase inhibitor MRK003 could significantly inhibit the sphere formation ability.

英文关键词： hepatocellular carcinoma; circulating tumor cells; tumor stem cells; biomarkers