基于分子印迹纳米复合体系的肝癌外周血中多肽标志物的筛选与鉴定

Early detection is an important aspect of efforts to improve survival in hepatocellular carcinoma(HCC).Molecular biomarkers indicative of physiologic state during HCC initiation and progression can provide increased sensitivity and specificity for diagnosis and classification of disease. The goal of this project is to discovery peptide biomarkers for HCC in peripheral blood. The project has three aspects: (1) Enrichment and separation of potential peptide biomarkers; (2) Analysis and identification of peptide biomarkers; (3) Assessment of peptide biomarkers for HCC in clinic. We address these issues through the development of a novel protocol that couples magnetic molecular imprinted nanomaterials (MIN) with MALDI-TOF-MS and bioinformatics for the identification of candidate biomarkers. The blood samples are collected from HCC and benign tumor of hepatocellular carcinoma. The different peaks in mass spectrum will be selected by comparing with those of different groups with CLINPROT? MALDI-TOF MS and bioinformatics technique. The selected potential peptide biomarkers act as template molecules in MIN. The MIN was prepared with surface molecularly imprinting on the surface of magnetic nanoparticles. These materials have double-function of enrichment and separation for target peptide. The MIN is used to capture specific peptides and deplete nonspecific peptides. Then, the peptides released from the MIN will be analyzed and identified with MALDI-TOF-MS and bioinformatics techniques. The peptides biomarker will be valued in clinic diagnose. This project can provide not only useful way for diagnose HCC but also new idea for sensitive detection biomarker.

早期发现和诊断是提高肝癌病人生存率的关键。实现肝癌早期诊断的一个有效途径就是特异性检测肝癌相关标志物。本项目将以筛选肝癌外周血中多肽标志物为目标,围绕肝癌外周血中低丰度的潜在多肽标志物的富集与分离，结构鉴定及其临床验证等三个关键问题，以原发性肝细胞癌（HCC）和肝良性肿瘤的外周血为诊断模型，通过液体芯片飞行时间质谱技术（CLINPROT? MALDI-TOF MS）与生物信息学技术检测分析不同诊断模型的蛋白多肽谱差异，筛选潜在多肽标志物。以这些多肽为模板分子，制备双功能（即选择性富集与磁场快速分离）的分子印迹纳米复合材料，实现外周血中目标多肽的富集与分离，并用MALDI-TOF-MS与生物信息学技术相结合对富集多肽进行分析鉴定，筛选出标志物并用于临床验证。该方法的发展不仅为肝癌的早期诊断提供有力工具，而且为标志物的高灵敏度检测提供新思路。

早期发现和诊断是提高肝癌病人生存率的关键。实现肝癌早期诊断的一个有效途径就是特异性检测肝癌相关标志物。在众多肝癌的致病原因中，最常见的为病毒性肝炎，尤其是乙型病毒（HBV）感染与肝癌的关系密切，HBV感染是原发性肝癌的首要病因。鉴于此，本项目以筛选HBV感染病人外周血中多肽标志物为目标，围绕HBV感染外周血中低丰度的潜在多肽标志物的富集与分离，结构鉴定及其临床验证等三个关键问题，以HBV感染病人外周血与健康人外周血为诊断模型，筛选出2882.89 Da （纤维蛋白原β链 FGB）与4476.12 Da （核磷蛋白亚型2 NPM）作为标志物。随后通过分子印迹、全内角反射荧光（TIRF）技术、超高效液相色谱与串联四级杆飞行时间质谱仪联用技术（UPLC/Q-TOF-MS/MS）对该标志物进行了检测。结果发现：分子印迹法受限于其选择性，难以用于临床样本的富集；而TIRF技术的检测限仍有待提高；UPLC/Q-TOF-MS/MS技术具有较高的检测灵敏度和选择性，可作为标志物的检测方法。本项目的开展不仅为肝癌的早期诊断提供有力工具，而且为标志物的高灵敏度检测提供新思路。