肝癌早期诊断脱氧核酶探针的筛选、靶标鉴定及作用机制研究

Hepatocellular carcinoma (HCC) is the primary malignancy in the liver with high mortality. Early diagnosis is the key to improve the survival rate of HCC patients, but how to monitor malignant transformation of hepatocytes and diagnose HCC early is still a medical problem. Currently, due to the poor sensitivity and specificity of traditional HCC detection methods, it is urgent to develop new recognition molecules and establish a new detection technology for HCC. In recent years, fluorogenic DNAzymes as the molecular probes have the advantages of strong specificity, high sensitivity and short detection time, but DNAzyme probes for early detection of HCC have not been reported. In our previous study, we used the extracellular mixtures of HCC cells as the intended targets for the SELEX experiment, and observed stable cleavage activity and good specificity of DNA pool after 14 rounds of SELEX selection. In this project, the 14th DNA pool will be cloned and sequenced, and several sequences with high homology will be selected as candidate probes. Then, the specificity and sensitivity of candidate probes will be detected and reaction conditions will be optimized. Subsequently, the core regions of the optimum DNAzyme probe will be determined by mutation and knockout, and its targets will be identified using proteomics and bioinformatics, and the interaction mechanism between DNAzyme probe and its target will be explored. This project will provide a new detection technology for early diagnosis of HCC, which has great value in clinical application.

肝细胞癌是肝脏中的首要恶性肿瘤疾病，死亡率较高。早期诊断是提高肝癌患者生存率的关键，但如何监测肝细胞恶性转化或早期诊断肝癌仍是医学难题。目前由于传统肝癌检测方法的灵敏度及特异性欠佳，急需发展新型识别分子，建立一种新的肝癌检测技术。近年来，荧光标记脱氧核酶作为分子探针，具有特异性强、灵敏度高、检测时间短等优势，但用于肝癌检测的脱氧核酶探针尚未见报道。本项目前期以肝癌细胞外液为混合靶标，通过14轮SELEX筛选，获得的DNA分子库已具有稳定的剪切率和良好的特异性。因此，本项目将首先对该分子库进行克隆测序，选取同源性较高的序列作为候选探针。然后对候选探针进行特异性检测、灵敏度分析和反应条件优化。最后用突变及敲除等方法确定效果最佳探针的核心区域，用蛋白质组学和生物信息学等方法对其靶标进行鉴定，进而探讨探针与靶标的相互作用机制。本项目将为肝癌早期诊断提供一种新的检测技术，具有重要的临床应用价值。

肝细胞癌是肝脏中的首要恶性肿瘤疾病，死亡率较高。早期诊断和治疗是提高肝癌患者生存率的关键。建立新颖的肝癌细胞检测技术，开发快速、灵敏度高、特异性强的肝癌检测试剂盒是亟待解决的重要课题之一。为解决该问题，本项目利用具有RNA切割功能的荧光标记脱氧核酶（RNA-cleaving fluorogenic DNAzymes，RFD）模型，建立了可特异性识别不同转移潜能人肝癌细胞的RFD分子探针检测技术。申请人以人肝癌细胞的蛋白提取液为混合靶标，采用RFD分子探针模型和指数式富集的配体系统进化（systematic evolution of ligands by exponential enrichment，SELEX）技术，通过多轮体外筛选，将最终富集到的文库进行克隆测序和一级序列同源性分析，再用RNA Structure、The mfold Web Server在线软件进行二级结构分析。根据丰度、同源性和二级结构，选取同源性较高的序列作为候选探针。将候选探针分别与人肝癌细胞的蛋白提取液作用，选取荧光强度较高的RFD分子探针用于后续分析。然后对获得的RFD分子探针进行了特异性检测、灵敏度分析和反应条件优化，获得了针对不同转移潜能肝癌细胞的高灵敏度、高特异性的RFD分子探针。利用蛋白酶解的方式证明其靶标为蛋白质，分子筛截留蛋白实验确定其靶标蛋白分子量大小范围。该研究为解决个体化用药生物标志物研发和建立一种快速、简便、低成本、高准确性的新型肝癌检测试剂盒提供了关键技术，以实现肿瘤的预防和早期诊断，具有重要的临床应用价值。