液相适配子阵列检测胃癌新技术的建立及其诊断和伺机性筛查价值评价

A simple and efficient method is critical for early diagnosis and treatment of gastric cancer. Gastroscopy and biopsy are efficient for the diagnosis of gastric cancer, but not simple, while the detection of serum tumor markers are simple but not efficient for the diagnosis of gastric cancer. The reasonable condition is that firstly screening out patients with high risk of gastric cancer by a simple and efficient method and then confirming the diagnosis by gastroscopy.Aptamers, generated from selection in vitro, are artificial nucleic acid ligands of biological molecules, which are like antibodies in function but with more advantages than antibodies and a good prospect for the diagnosis of diseases. Previously, we selected 86 aptamers against gastric cancer (GC) serum and preliminaryly evaluated 7 of them, and found that they are valuable for the diagnosis of GC, especially the combination of 3 aptamers with independent diagnostic value for gastric cancer with accuracy 90.2%. We speculated that the combined application of these aptamers could be efficient for GC diagnosis . In the present study, we will select a group of GC aptamers with independent diagnostic value for gastric cancer from the 86 aptamers and develop novel liquid aptamer arrays for detection of gastric cancer, and prospectively evaluate their value in diagnosis and opportunistic screening of gastric cancer. It is anticipated that this study will create a novel method for gastric cancer diagnosis and screening, and meanwhile expand our work about serum aptamer selection and cancer diagnosis previously supported by the National “863” Project and National Science Foundation of China.

既简便又有效的诊断技术是目前提高胃癌早诊早治水平的关键。胃镜检查加活检诊断胃癌有效但不简便，常用血清肿瘤标志物检测诊断胃癌简便但无效。理想状态是先用简便有效的方法筛选出胃癌高危患者，再胃镜检查明确诊断。适配子是生物分子的人工核酸配体，由体外筛选获得，功能类似抗体，但具有热稳定等优势，诊断应用前景良好。我们前期筛选到86个胃癌血清适配子，初步分析的7个适配子对胃癌诊断价值良好，3个有独立诊断价值的适配子联合检测诊断胃癌的准确度达90.2%。我们推测胃癌血清适配子联合检测可以有效地诊断胃癌。为此，本研究将从这86个适配子筛选出一组对胃癌具有独立诊断价值的适配子，创建液相适配子阵列检测胃癌新技术，并前瞻性评价其对胃癌的诊断和伺机性筛查价值，为开辟胃癌诊断和筛查新途径奠定基础。本研究将取得原创性成果，并进一步深化我们前期在863项目和国家基金项目资助下开展的血清适配子筛选与肿瘤诊断研究方面的工作。

目前临床亟需简便有效的方法筛选出胃癌高危患者，再进行胃镜检查明确诊断。核酸适配子是生物分子的人工单链寡核苷酸配体，被认为有重要诊断应用前景。本项目在前期工作基础上，创建液相适配子阵列检测胃癌新技术，并前瞻性评价其诊断和伺机性筛查胃癌的价值，旨在为开辟简便有效的胃癌筛查诊断新途径奠定基础。.本项目按研究计划执行，达到了预期的研究目标。.（1）采用聚丙烯酰胺凝胶分析，从前期筛选到的86个胃癌血清适配子中优选出11个代表性适配子，它们与胃癌血清的结合明显强于良性胃病血清，结合胃癌血清的Kd值为0.04~0.62μM，表明有良好的特异性和亲和力。.（2）在全面优化实验条件后用11个代表性适配子分别检测胃癌、良性胃病和正常对照血清标本各192例的三重荧光，后者鉴别胃癌和非胃癌的受试者工作特征曲线下面积（AUROC）达0.687~0.832，表明对胃癌有较好的诊断价值。.（3）对各适配子的三重荧光指标进行双指标相关分析，以相关性差（诊断价值独立）的2~3个适配子组合，成功构建出13个适配子阵列，它们鉴别胃癌和非胃癌的AUROC为0.752~0.905，其中诊断价值最大一个适配子阵列进行了大样本血清标本验证分析（胃癌608例，良性胃病544例），鉴别胃癌与良性胃病的AUROC为0.815，仍然良好。.（4）对上述最佳适配子阵列以接受胃镜检查的372例（胃癌191例、良性胃病181例）患者进行前瞻性评价，显示鉴别胃癌与良性胃病的AUROC为0.871，敏感度为80.6%，特异度为78.6%，准确度为79.6%，达到良好水平，表明对胃癌有良好的伺机性筛查价值。.（5）对上述接受胃镜检查患者的传统血清肿瘤标志物和常规临床指标进行了胃癌伺机性筛查价值的分析，发现CEA、糖化血清蛋白（GSP）和血Ca2+对胃癌有一定的筛查诊断价值，AUROC分别为0.724、0.725和0.677，另以7项血液检验指标建模筛查诊断胃癌AUROC达0.917。这些基于临床资料的发现值得进一步研究。.（6）综合上述胃癌血清适配子阵列和临床指标的分析结果，提出了胃癌伺机性筛查流程，并绘制流程图，值得在后续研究工作中进一步认证。.综上所述，本项目的研究成果不但为建立胃癌诊断和伺机性筛查新方法提供了依据，也为核酸适配子的临床应用和常规临床数据的价值挖掘提供了新思路。