基于DNA组装技术比色检测肝癌标志物外泌体的新方法研究

Hepatocellular carcinoma-derived exosomes can promote the growth, migration and invasion of hepatoma cells, and are related to various exosomal miRNAs abnormally expression, indicating hepatocellular carcinoma-derived exosomes is a new biomarker for the diagnosis and prognosis of liver cancer, which possess a wide prospect of clinical application. However, for the low concentration of exosomes in the early stage of cancer, reliable and convenient quantitative methods of exosomes are still technically challenging. Taking hepatocellular carcinoma-derived exosomes for research objects, this study designed LZH8 aptamer elaborately, and combined the specific recognition of LZH8 aptamer to hepatocellular carcinoma-derived exosomes with DNA assembly signal amplification strategy, and then introduce a probe which can produce a colorimetric signal, such as enzyme modified with DNA sequence or DNA mimic enzyme formed by DNA assembly. Next, to obtain high sensitivity, the reaction conditions and the activity of DNAzyme were optimized. Finally, a series of simple colorimetric detection strategies for hepatocellular carcinoma-derived exosomes detection with high sensitivity and specificity were constructed. These strategies provided clinical value for early diagnosis, prognosis and monitoring of liver cancer.

肝癌细胞分泌的外泌体能促进肝癌细胞的生长及迁移侵袭，且其涉及多种外泌体miRNA的异常表达，是一种新的肝癌诊断和预后生物标志物，在临床上具有广阔的应用前景。然而，在癌症早期阶段，外周血中肝癌细胞外泌体含量较低，发展简单且灵敏快速的检测方法一直面临着巨大挑战。本项目以肝癌细胞来源的外泌体为研究对象，通过对特异性识别肝癌外泌体的LZH8适配体进行巧妙设计，进而将LZH8适配体对外泌体的识别作用和DNA组装信号放大策略有机结合在一起，在此基础上引入可产生比色信号的探针，如修饰DNA的酶探针或通过DNA组装直接形成高催化活性的DNA酶作为探针等，将适配体对外泌体的识别转换成肉眼可见的颜色输出，接着通过对反应条件进行优化、探索如何提高DNA模拟酶的催化活性等，最终建立一系列高灵敏、高特异性且简单快速肝癌外泌体比色检测策略，实现对肝癌患者外泌体的检测，为肝癌的早期诊断、预后及监测等方面提供临床价值。

肿瘤外泌体能够较好的反映母细胞特性，有望成为肿瘤诊断和预后的标志物。然而肿瘤早期阶段，肿瘤细胞来源的外泌体含量较低，发展高灵敏的外泌体定量检测策略，具有十分重要的意义。基于DNA组装信号放大技术为外泌体高灵敏检测提供了便捷。本研究以肿瘤细胞来源的外泌体为研究对象，将适配体对外泌体的识别作用和DNA组装信号放大策略有机结合在一起，并把这种识别作用转换成可视化、电致化学发光等可检测信号，发展了多种新型外泌体的检测策略，主要包括以下几个方面：（1）通过DNA组装技术构建DNA微胶囊，将乙酰胆碱酯酶包裹在内部，外泌体表面蛋白CD63结合DNA胶囊上的适配体，破坏胶囊结构，释放出乙酰胆碱酯酶，催化底物改变体系的pH，使酚红变色，实现对外泌体的可视化检测。（2）将硫化镉量子点包裹在DNA胶囊中，利用外泌体的识别破坏DNA胶囊，在内切酶的作用下，进行循环反应，释放出量子点，利用量子点的电致化学发光信号对外泌体检测。（3）在外泌体表面修饰DNA探针，将其和金纳米粒子修饰的核酸结合在一起，构建DNA“步行者”机器，利用邻位效应形成DNA酶，对金纳米粒子上修饰的核酸切割，释放出的单链片段和修饰在电极上的DNA进行杂交，利用亚甲基蓝的电化学信号对外泌体进行检测。（4）构建外泌体响应的DNA信号放大反应，分离出反应产物，和修饰在电极表面的DNA序列构建“步行者”机器，采用双重信号放大实现对外泌体的检测。（5）在电极表面修饰核酸，利用端粒酶对引物的延伸作用，从而诱发DNA的邻位反应，并利用内切酶进行信号放大，实现了对肿瘤标志物端粒酶的检测。这些研究建立一系列高灵敏、高特异性且简单快速的外泌体检测策略，实现对肿瘤患者外泌体的检测，为疾病的早期诊断、预后及监测等方面提供重要的临床价值。