特异性适配体生物传感器靶向诊断肝癌的新技术研究

Hepatoma is one of the most common malignant tumors which severely threatens human health and life. Therefore, it is essential to discover some new ideal strategy of diagnosis and treatment with high sensitivity and specificity for hepatoma. In this project, the aptamers against hepatoma carcinoma markers AFP、GP73、DKK-1 and Endoglin were selected on the basis of the corresponding aptamers selection cell models prepared by CRISPR-Cas system with strong penetration of small molecule peptide guide, which was constructed by our research group. Then, the aptamers, which will be combined with nanomaterials thereby possessing the effect of signal-amplification or signal-tracing, will be used as probes to identify AFP、GP73、DKK-1 and Endoglin. Based on the technique, three sub-techniques will be developed. To be exactly, the graphene/hemin nano-probe will be developed to detect specifically AFP、GP73、DKK-1 and Endoglin in serum. Secondly, multi-target fluorescent detection will be used to detect in tissue specimen via the development of fluorescent aptamer nano-probe. Thirdly, the development of magnetic aptamer nano-probe will allow the research on diagnosis of hepatoma via in vivo hepatoma-targeted magnetic resonance imaging (MRI) to be achieved. Therefore, this study shows great possibility for improving sensitivity and specificity of diagnostic methods to increase detection rate of hepatoma. Furthermore, the research will provide new methods and ideas for targeting diagnosis of hepatoma.

肝癌是临床上最常见恶性肿瘤之一，严重威胁人类健康和生命。寻找特异性与灵敏度更理想的肝癌诊断新技术是目前临床肝癌诊断研究领域的重点和难点。本项目拟以肝癌标志物AFP、GP73、DKK-1、Endoglin为靶点，利用本研究团队协同创新建立的高穿透力小分子引导肽CRISPR-Cas细胞基因编辑技术构建筛选适配体的细胞模型并筛选出相应的适配体。以适配体为识别探针，结合纳米材料的信号放大作用或示踪效应，构建石墨烯/血红素适配体纳米靶向探针，在血清学水平建立针对肝癌的多指标联合检测生物传感技术平台；构建荧光适配体纳米靶向探针，在病理组织学水平进行多指标组织标本荧光检测；构建磁性适配体纳米靶向探针，在影像学水平开展体内靶向肝癌磁共振成像诊断技术研究。本研究有望增强肝癌诊断方法的灵敏度和特异性，提高早期肝癌检出率，为肝癌的靶向诊断提供新思路，开辟新途径。

项目组在本项目实施期间，开展了以下研究：.1. 在适配体筛选方面，将CRISPR/Cas9技术与细胞SELEX筛选技术结合，筛选出具有更高特异性和亲和力的AFP、GP73、DKK-1、Endoglin等适配体。..2. 在适配体纳米靶向探针构建方面，制备出能特异结合肝癌新生肿瘤血管内皮细胞的磁性纳米探针用于肿瘤磁共振成像研究；制备出能高灵敏识别肿瘤细胞的适配体荧光硅纳米检测探针，实现对肿瘤的靶向诊断。 ..3. 在适配体传感器用于肿瘤标志物检测方面，创建了经磁富集和滚环扩增双信号放大的高特异、高灵敏肿瘤智能检测系统；构建了适配体修饰石墨烯/血红素的类过氧化物酶放大检测肿瘤标志物的适配体生物传感器；利用HRP标记的羊抗人IgG包裹纳米金制备出稳定性强、分散性好、能增强分子靶向性的信号放大免疫探针，可高灵敏检测肝癌等肝病患者及正常人血清中抗AFP、GP73、DKK-1、Endoglin抗体的水平，为肝癌等恶性肿瘤的诊断提供新思路。..4. 在恶性肿瘤关键新靶点研究方面，首次证明盐诱导激酶1为肝癌发展中关键抑癌基因；开发出分离高纯度Endoglin（CD105）阳性内皮细胞的策略，通过比较肿瘤新生血管内皮细胞和正常内皮细胞中蛋白表达的差异，发现神经纤维蛋白1和血小板衍生的生长因子受体β有望成为抗血管生成治疗的潜在生物标志物。..本项目共发表SCI论文12篇；获国家授权发明专利3项，国家受理发明专利12项；培养中组部“万人计划”2名，科技部“中青年科技创新领军人才”2名，国家“百千万人才工程”国家级人选2名，国家有突出贡献中青年专家2名，国务院政府特殊津贴专家2名；培养博士后2名，博士4名，硕士33名；获国家教育部自然科学奖一等奖1项；积极开展学术交流，项目组共举办学术会议6次，先后参加国际学术会议11次。