基于适配子组三重血清荧光高通量测定建立“一站式”肝癌诊断新技术及其前瞻性评价

Early diagnosis, accurate staging and liver function grading are crucial for improving the therapeutic effect and prognosis of patients with primary hepatic carcinoma (PHC), which are based on complete information of clinic, laboratory, imageology and pathology. Therefore, it is important to investigate a “one-stop” method to simultaneously diagnosis PHCs (including early, small and AFP-negative PHCs), stage and grade liver function through one test. Aptamers are artificial nucleic acid ligands of biological molecules, which are functionally like antibodies, but with more advantages. Previously, we generated 365 aptamers against PHC serum and developed a novel high throughput diagnostic method based on aptamer-related triple serum fluorescence. With this method, we found that the combination analyses of some aptamers in a large sample size were useful for PHC diagnosis, staging and liver function grading. So we speculate that a "one-stop" diagnostic method for PHC can be achieved by high-throughput detection and combination analysis of triple serum fluorescence of a group of aptamers that can complement with each other. In the present study, we will develop a “one-stop” PHC diagnostic method by high throughput detection of triple serum fluorescence of a group of aptamers selected from previously generated aptamers and evaluate its diagnostic value prospectively. This study is consistent with the modern concept of experimental diagnostics, high-throughput detection followed by combination analysis, which will promote the updating of PHC diagnostic techniques and systematize our research in the field of aptamers.

尽早诊断、准确分期和肝功能分级是提高肝癌疗效和改善预后的关键，但其完成需要完善的临床、实验室、影像学和病理资料，探索集肝癌诊断（包括早期肝癌、小肝癌和AFP阴性肝癌）、分期和肝功能分级于一体的“一站式”诊断新技术有重要意义。适配子是生物分子的人工核酸配体，功能上类似抗体，但更具应用优势。前期我们筛选到365个肝癌血清适配子，创建了适配子三重血清荧光高通量测定技术，大样本分析发现适配子三重血清荧光联合建模对肝癌有良好的诊断价值和一定的分期及肝功能分级价值。我们推测高通量检测一组诊断价值互补的适配子的三重血清荧光并建立诊断模型，可实现肝癌“一站式”诊断。为此，本研究将优选前期筛选到的适配子构建适配子组，高通量检测适配子组三重血清荧光，创建肝癌“一站式”诊断新技术，并前瞻性评价其诊断价值。本研究契合高通量联合检测的现代实验诊断新理念，将助推肝癌诊断技术升级换代及系统化我们在适配子领域的研究工作。

目前临床亟需集肝癌诊断（包括早期肝癌、小肝癌和AFP阴性肝癌）、分期和肝功能分级于一体的“一站式”诊断新技术。适配子是生物分子的人工核酸配体，被认为有重要诊断应用前景。本项目在前期工作基础上，优选适配子并构建成肝癌“一站式”适配子组，高通量检测适配子组三重血清荧光，创建肝癌“一站式”诊断新技术，并前瞻性评价其对肝癌诊断、分期和肝功能分级的价值，为推动肝癌诊断技术升级换代和诊疗水平的提高奠定基础。.本项目按研究计划执行，达到了预期的研究目标。.（1）采用Clustal X和RNA Structure4.6软件从前期筛选到的365个肝癌血清适配子中优选出26个二级结构独特和序列同源性低的代表性肝癌血清适配子。.（2）先采用肝癌和肝硬化混合血清逐一分析了26个代表性肝癌血清适配子，再采用单个血清小样本分析其中14个高特异性的肝癌血清适配子在不同实验条件下“一站式”诊断肝癌的价值，再对其中6个 “一站式”诊断价值大的适配子扩大样本分析，最终优选出4个对肝癌“一站式”诊断价值大的适配子。.（3）采用SPSS Modeler 18.0软件对4个优选肝癌血清适配子的荧光指标进行多因素建模分析，发现支持向量机和C5.0决策树模型构建的“一站式”诊断适配子组对肝癌“一站式”诊断价值最大，这两种模型诊断肝癌（早、中、晚期肝癌、小肝癌、AFP 阴性肝癌和全部肝癌）的AUROC为0.943~1.000；进行BCLC 分期、TNM 分期和肝功能分级的准确率分别为82.9~89.3%、87.2~94.8%和83.3~96.1%。.（4）将540例前瞻血清标本的三重血清荧光代入相应模型，结果仍然显示支持向量机和C5.0决策树模型构建的“一站式”诊断适配子组对肝癌“一站式”诊断效果最佳，这两种模型诊断肝癌（早、中、晚期肝癌、小肝癌、AFP 阴性肝癌和全部肝癌）的AUROC为0.846~1.000；进行BCLC 分期、TNM 分期和肝功能分级的准确率分别为74.6~79.6%、84.7~95.1%和79.3~97.8%。.综上所述，本项目成功创建了基于适配子组三重血清荧光的肝癌“一站式”诊断新技术，达到了一次血清检测完成肝癌诊断、分期和肝功能分级的目的。