糖胺聚糖结合分子群预测肝癌复发转移和预后及其机制探讨

Glycosaminoglycans (GAGs) are large complex carbohydrate molecules （e.g. heparin） that interact with a wide range of proteins involved in physiological and pathological processes. A significant proportion of cytokines belong to GAG-binding cytokines, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), midkine, and osteopontin (OPN). All of them are present in the tumor microenvironment, promoting tumor growth, invasion and/or angiogenesis. Serum levels of these molecules are frequently elevated in patients with hepatocellular carcinoma (HCC), and usually correlated with the metastasis and a poor prognosis. However, single molecule detection has low sensitivity, poor specificity, and limited clinical value, thus it is not used in the routine clinical laboratory so far. A quantitative assay for the total content of multiple molecules in the theory will not only retain their original information, but also improve the sensitivity and specificity by cumulative contents of differential expression of all these molecules. Based on heparin-antibody sandwich principle, this project will develop a novel chemiluminescence immunoassay method, which could determine the total content of above-mentioned five molecules. Through a systematic methodology, we will evaluate the accuracy, reproducibility, and feasibility of this assay for the total content of multiple molecules. Furthermore, we will perform large-scale testing of clinical blood samples by using the new method, and investigate the role of the total content of the five molecules in the prediction of the recurrence, metastasis, and prognosis in HCC and other types of tumors. Finally, we will focus on identification of the molecular mechanisms by which a combination of glycosaminoglycan binding molecules promote invasion and metastasis through inducing epithelial-mesenchymal transition (EMT) in HCC. This study is expected to provide a new means to help improve diagnosis and treatment of HCC, and develop a new research mode for "salving a complex clinical diagnostic problem by detection of a pool of molecules".

碱性成纤维细胞生长因子、血管内皮生长因子、肝细胞生长因子、中期因子和骨桥蛋白等糖胺聚糖（肝素）结合因子在肝癌血清中含量通常较高，在预测肝癌复发转移和预后方面具有不同程度的潜能。但是，单分子检测灵敏度低、特异性差，临床价值有限，因而尚不能在临床常规应用。而多个分子总量检测在理论上不仅会保留各自原有的信息，而且会通过叠加各个分子的表达差异，提高灵敏度和特异性。因此，本项目根据肝素-抗体双夹心法原理，创建上述5个分子群总量的化学发光免疫测定方法。通过系统的方法学研究，评价多分子总量检测的准确性、重复性和可行性；通过临床应用研究，评价新检测方法预测肝癌和其他肿瘤复发转移和预后的应用价值；通过相关机制研究，鉴定组合糖胺聚糖结合因子通过诱导上皮间质转化促进肝癌细胞侵袭转移的分子信号通路。预期本项目可提供一个有助于提高肝癌诊治水平的新手段，并且发展一个"以某一分子群诠释一个临床问题"的新研究模式。

肝细胞癌（hepatocellular carcinoma，HCC）是我国最常见的恶性肿瘤之一。已知一些糖胺聚糖（肝素）结合因子在肝癌血清中含量通常较高，在预测肝癌复发转移和预后方面具有不同程度的潜能。本项目试图创建一种糖胺聚糖（肝素）结合因子分子群总量的化学发光免疫测定方法，用于预测肝癌复发转移和预后。主要研究内容包括通过系统的方法学研究，评价多分子总量检测的准确性、重复性和可行性；通过临床应用研究，评价新检测方法预测肝癌和其他肿瘤复发转移和预后的应用价值；通过相关机制研究，鉴定糖胺聚糖结合因子诱导上皮间质转化、促进肝癌细胞侵袭转移的分子信号通路。.围绕本课题主要内容，我们开展了以下研究。.在方法学研究方面，采用抗体-肝素双夹心ELISA法检测肝素结合因子碱性成纤维细胞生长因子（basic fibroblast growth factors，bFGF）、血管内皮生长因子（vascular endothelial growth factor，VEGF）、肝细胞生长因子（hepatocyte growth factor，HGF）和中期因子（midkine，MK）等4种肝素结合因子总量，采用双抗体夹心的电化学发光免疫法检测HGF、MK和血小板衍生生长因子（platelet-derived growth factor，PDGF）等3种因子总量，发现后一种方法更稳定，更敏感，可现性更好。检测结果显示，肝癌组与良性肝病组及正常人组之间3种因子总量具有显著差异（P < 0.05），有助于肝癌诊断（ROC曲线下面积为0.8）。但是肝癌组与其他类型肿瘤组之间无显著差异（P > 0.05）。肝癌患者术后血清3种因子总量持续下降，而良性肝病患者术后下降则不明显。.在机制研究方面，我们重点研究了中期因子参与促进肝癌循环肿瘤细胞（circulating tumor cells，CTCs）形成和失巢凋亡抵抗的分子机制，包括验证了MK诱导肝癌细胞抵抗失巢凋亡的设想，采用多种技术手段，验证和鉴定信号转导过程，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，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。.在项目实施过程中，主要培养了2名研究生，发表了1篇项目标注中文核心期刊论文，2篇项目标注SCI期刊论