基于表面工程的外泌体digital PCR定量分析体系的构建及转化医学研究

最近以来，肿瘤外泌体被发现在肿瘤转移前微环境形成和肿瘤细胞转移的过程中发挥重大作用。发展一种外泌体定量分析方法，无论对于外泌体的科学研究还是相关疾病的诊断都具有重要的意义。本项目通过表面工程技术修饰外泌体然后利用数字PCR方法对外泌体进行定量分析。首先通过生物锚定分子（biocompatible anchor molecule，BAM）将DNA链锚定在外泌体表面，标记后的外泌体利用数字PCR芯片进行定量分析，通过PCR检测外泌体表面的DNA实现外泌体定量分析。肿瘤特异性外泌体定量分析则通过抗体-DNA和BAM-DNA双标记实现。外泌体经抗体-DNA和BAM-DNA双标记后进行邻位连接反应，只有肿瘤特异性外泌体上能标记抗体-DNA和进行邻位连接反应，形成新的DNA，带有新DNA的外泌体通过数字PCR芯片定量分析。两种外泌体定量分析方法的建立将为肿瘤的研究和诊断提供帮助。

Recently, reports showed that tumor exosome exerted major influences in metastatic niche formation and organ-specific metastatic. It is a meaningful work to develop a quantitative analysis method of exosome for scientific research and related disease diagnosis. This proposal intent to quantitatively analyze amount of exosome by digital PCR after exosomes modified with surface engineering technology. External DNA chains were anchored on the surface of exosomes by biocompatible anchor molecule (BAM). Labelled exosomes were quantitatively analyzed by digital PCR chip with amplifying the DNA on exosomes with PCR. Tumor specific exosome quantitative analysis was realized with double-labelling of antibody-DNA and BAM-DNA. When tumor specific exosomes were double-labelled with antibody-DNA and BAM-DNA, proximity-dependent DNA ligation assay was carried out. But only the tumor specific exosomes can been double-labelled and accomplish proximity-dependent DNA ligation assay to generate a new DNA chain. The tumor specific exosomes were quantitatively analyzed by digital PCR chip with new DNA chain on exosomes. Such methods could aid scientific research and therapy decision of cancer.