循环肝癌细胞形成和抵抗失巢凋亡的分子机制和临床意义研究

With the advent of new analytical technologies, both basic scientific and translational research on circulating tumor cells (CTCs) is advancing rapidly. However, probably due to current commercial EpCAM-based methods unavailable for capturing hepatocellular carcinoma (HCC) cells, few studies examining CTC counts have been conducted on HCC patients so far. Recently, we developed a novel detection system for HCC CTCs (asialoglycoprotein receptor-based magnetic cell separation combined with Hep Par 1 staining detection). The study results suggest that it would be of great value not only to enumerate CTCs but also to characterize them with the ability to invade, proliferate, and cause a metastasis. Unfortunately, little is known about the biology of CTCs in HCC. Based on our results and the existing literature, we propose a hypothesis for the regulation mechanisms of CTC formation and survival that midkine (MK), a cytokine present in HCC cells and tumor microenvironment, activate the PI3K/AKT pathway via tyrosine kinase receptor B, and subsequently upregulate the transcription factor ZEB1, mediating epithelial-mesenchymal transition (EMT) in cancer cells, which results in the acquisition of more invasive and stem-like traits, and have an increased ability to migrate into the bloodstream and display more resistance to anoikis. In addition to collection of further experimental evidence for MK-induced EMT and anoikis resistance, this project will focus on hypothesis verification by using neutralizing antibodies, signalling pathway specific inhibitors, and small interfering RNA to shut down a signaling pathway, finally figuring out how MK mediates EMT and the anoikis resistance by upregulation of ZEB1 in HCC. Moreover, we will molecularly characterize CTCs and investigate its clinical relevance, developing a new model for personalized diagnosis and therapy of HCC based on multiplex assays for the markers of EMT and anoikis resistance of HCC CTCs.

最近我们创建了一种独特的不依赖EpCAM的肝癌CTCs分离/检测系统。研究结果提示，检测少数更具存活力和侵袭性的CTCs亚群比单独CTCs计数可能更有价值。然而，目前对于肝癌CTCs的形成过程和存活调节机制尚不清楚。根据前期研究结果和现有研究资料，我们推测中期因子（MK）通过上调或激活酪氨酸激酶受体B，活化PI3K/AKT信号通路和/或Notch通路，上调转录因子ZEB1，介导肝癌细胞EMT，进而促进肝癌CTCs形成和失巢凋亡抵抗。本项目将在完善MK诱导肝癌EMT和失巢凋亡抵抗的实验证据的基础上，重点研究推测的作用机制。计划采用中和性抗体、信号通路特异性抑制剂、微小RNA干扰等多种技术手段，详细验证和鉴定信号转导过程，描绘出MK上调ZEB1介导肝癌细胞EMT和失巢凋亡抑制的完整信号转导通路。同时通过相关的临床研究，提出一个基于肝癌CTCs EMT和失巢凋亡标志物检测的肝癌个体化诊疗新模式。

肝细胞癌（hepatocellular carcinoma，HCC，以下简称肝癌）复发转移率很高，循环肿瘤细胞（circulating tumor cells，CTCs）已被认为是其主要根源。根据前期研究结果和现有研究资料，我们推测中期因子（midkine，MK）参与促进肝癌CTCs形成和失巢凋亡抵抗。本项目的主要内容是在完善MK诱导肝癌上皮间质转化（epithelial-mesenchymal transition，EMT）和失巢凋亡抵抗的实验证据的基础上，重点研究推测的作用机制，包括采用中和性抗体、信号通路特异性抑制剂、微小RNA干扰等多种技术手段，验证和鉴定信号转导过程，描绘出MK介导肝癌细胞失巢凋亡抑制的完整信号转导通路。同时通过相关的临床研究，提出一个基于肝癌CTCs失巢凋亡标志物检测的肝癌个体化诊疗新模式。.围绕本课题主要内容，我们开展了以下研究。.改进了肝癌CTCs分离/检测方法。一是建立基于ASGPR、氨甲酰磷酸合成酶1（carbamyl phosphate synthetase 1，CPS1）和广谱细胞角蛋白（cytokeratins，CKs）等三抗体的肝癌CTCs检测方法。二是建立基于阴性选择的ASGPR和CPS1等二抗体的肝癌CTCs检测方法。这些改良方法减少了假阳性及假阴性结果，进一步提高了检测效率。三是建立了基于微流控芯片的肝癌CTCs捕获与计数的方法，简化了检测步骤，缩短了检测时间，减少了样本采集量，并且提高了检测敏感性和特异性。微流控芯片捕获的肝癌CTCs释放后可以进行培养和药敏实验。这些方法为进一步研究肝癌CTCs的生物学特性奠定了实验基础。.构建了肝癌CTCs肝转移动物模型，为进一步研究肝癌CTCs在肝癌复发转移过程中的作用提供了重要的实验动物平台。.验证了MK诱导肝癌细胞抵抗失巢凋亡的设想。.发现了MK通过表面受体间变性淋巴瘤激酶（Anaplasticlymphoma kinase，ALK）调节肝癌细胞失巢凋亡的现象。.阐述了结直肠癌细胞抵抗失巢凋亡的分子机制，找到了在肠癌复发、转移过程中发挥重要作用的关键因子，为防治结直肠复发、转移提供新的思路。.在项目实施过程中，主要培养了4名研究生，发表了7篇项目标注中文核心期刊论文，8篇项目标注SCI期刊论文，参加了1次国际会议交流，申请了1项发明专利。