基于适配子的AFP阴性肝癌新血清标志物筛选及前瞻性验证

The diagnosis of alpha-fetoprotein negative primary hepatic carcinoma (ANHC) is a big challenge. Discovery of new sensitive and specific serum biomarkers is ideal strategy to address this problem. Aptamers, generated via systematic evolution of ligands by exponential enrichment, are artificial nucleic acid ligands of biological molecules. aptamers are like antibodies in recognition and binding of target molecules, but superior to antibodies in preparation and hot stability. Selection of aptamers against serum followed by capturing and analyzing their target proteins in serum is a simply and efficiently new strategy to discover serum biomarkers. We previously selected a group aptamers against ANHC serum, and three of them were highly valuable for diagnosing ANHC, which were validated in a large size of serum samples. We speculate that the target proteins of the three aptamers are possible serum biomarkers of ANHC. In this study, we will capture their target proteins in serum and analyze them by qualitative mass spectrometry to find unique or elevated target proteins in ANHC serum, and then validate them via MRM quantitative mass spectrometry in serum specimens to select and validate candidate biomarkers of ANHC. Finally, we will apply ELISA method to prospectively measure these candidate biomarkers in a large size of serum specimens and evaluate their diagnostic value for ANHC for determining high sensitive and specific new ANHC serum biomarkers. It is anticipated that this study will provide original findings, and meanwhile systematize our work in the field of aptamers and hepatic carcinoma diagnosis.

AFP阴性肝癌（ANHC）是肝癌诊断中的难点，找到敏感而特异的ANHC血清标志物是理想的解决策略。适配子是生物分子的人工核酸配体，通过指数富集的系统进化技术筛选获得，其靶分子识别和结合功能可媲美抗体，但在制备和热稳定方面优于抗体。筛选血清适配子进而捕获和分析其血清靶蛋白，是简便高效发现血清标志物的新策略。我们前期筛选到一批AFP阴性小肝癌血清适配子，其中3个经大样本分析证实诊断ANHC价值良好，推测其血清靶蛋白可能是ANHC特异性标志物。本研究将捕获这3个适配子的血清靶蛋白并进行定性质谱分析，找出ANHC特有或高表达蛋白，通过MRM定量质谱测定其血清含量，验证并优选出ANHC候选标志物，前瞻性大样本ELISA检测并评价候选标志物对ANHC的诊断价值，找出敏感而特异的新标志物。本研究有望取得原创性研究成果，为创建ANHC诊断新方法奠定基础，并进一步系统化我们在适配子与肝癌诊断领域的研究工作。

AFP阴性肝癌是肝癌诊断中的难点，找到敏感而特异的AFP阴性肝癌血清标志物是理想的解决策略。筛选血清适配子进而捕获和分析其血清靶蛋白，是简便有效发现血清标志物的新策略。我们前期筛选到一批AFP阴性小肝癌血清适配子，其中3个经大样本分析证实对AFP阴性肝癌有良好诊断价值。本研究将对这3个适配子的血清靶蛋白进行捕获和分析，寻找AFP阴性肝癌的血清新标志物并进行前瞻性评价。..以上述3个适配子为捕获分子，通过磁珠法成功捕获到它们的血清靶蛋白，经定性质谱分析，其中有45个蛋白只在AFP阴性肝癌血清中被捕获到，而在良性肝病和正常对照血清中未捕获到，其中33个蛋白与肝癌或肿瘤相关。..从捕获的靶蛋白中优选出19个进行多反应监测（MRM）质谱分析，发现C4BPA、AACT、A1AG1、SAA1、HPT、HPTR等血清靶蛋白在AFP阴性肝癌血清中表达水平高于良性肝病1.5倍以上，区分AFP阴性肝癌与良性肝病的受试者工作特征曲线下面积（AUROC）为0.700~0.788。ELISA法测定结果显示C4BPA和 SAA1在AFP阴性肝癌的血清水平高于良性肝病，区分两者的AUROC为0.698~0.896，但不及异常凝血酶原（DCP）。..为寻找更佳的AFP阴性肝癌血清新标志物，进一步采用DIA质谱法进行血清蛋白组学分析并发现了102个AFP阴性小肝癌与对照间的差异表达蛋白，优选其中19个进行MRM质谱测定它们的血清水平，发现LRG1、HC2等少数几个蛋白在AFP阴性肝癌与良性肝病间差异表达较明显，有一定的诊断价值。..综合分析上述结果，优选出8个AFP阴性肝癌候选血清标志物进行前瞻性评价，ELISA法测定它们在肝癌和对照血清标本中的水平。发现A1AG1、LRG1、HPT、SAA2和C4BPA区分AFP阴性肝癌与对照具有良好或较大价值，AUROC为0.749-0.955，优于或相当于DCP（AUROC=0.759），各标志物的血清水平在AFP阴性和AFP阳性肝癌间无明显差异，诊断价值优于或相当于AFP。..本项目建立了基于适配子策略的AFP阴性肝癌血清标志物筛选和验证方法，成功筛选到有诊断价值的血清标志物，为建立AFP阴性肝癌，包括AFP阳性肝癌诊断新方法奠定了良好基础，也为疾病新血清标志物筛选研究提供了具有通用价值的新策略。