仿病毒融合囊泡快速原位检测肿瘤外泌体内miRNA

Exosomal miRNAs are important indicators for tumor diagnosis and prognosis. Despite budding methods to detect exosomal miRNAs recently, the problems of low recovery rate and huge consumption of time remain unsolved. In addition, exosomes have to be destroyed during miRNAs detection, resulting in the loss of miRNAs and making it difficult to ensure the accuracy of the results. Therefore, it is urgent to establish a method that can detect exosomal miRNAs rapidly and accurately. Inspired by the active cell membrane invasion of virus, this study aims to establish virus-mimetic fusion vesicles with the envelope protein of human parainfluenza virus 3 on the surface. These proteins are capable of binding to the sialic acid receptor on the surface of the exosomes, inducing efficient fusion between vesicle membrane and exosome membrane. Then the molecular beacons within the vesicles can be delivered into the exosomes. After binding to the exosomal miRNAs, the fluorescence signal of molecular beacon switches from the "closed" to "open" state, realizing in situ, rapid and accurate detection of exosomal miRNAs. This method avoids the destruction of the exosomes and the degradation of exosomal miRNAs by the nuclease in body fluid. In addition, viral fusion protein-mediated active fusion is more efficient and the detection efficiency is higher. This project provides a new tool for the detection of exosomal miRNAs and is of great significance for the early diagnosis, curative evaluation and prognosis analysis of cancer.

外泌体内miRNA可以作为肿瘤诊断的重要标志物。近年来检测外泌体内miRNA的方法层出不穷，但多存在耗时、回收率低的问题。此外提取miRNA时还需破坏外泌体，造成miRNA的损失并影响结果准确性。因此亟需建立一种可以快速准确检测外泌体内miRNA的方法。受病毒融合细胞膜入侵细胞的启发，本项目拟构建一种仿病毒融合囊泡。囊泡表面具有人副流感病毒3的包膜蛋白，该蛋白通过结合外泌体表面唾液酸受体引起囊泡与外泌体的高效融合，从而将囊泡内的分子信标递送进入外泌体。与外泌体内特异miRNA结合后，分子信标荧光信号开启，从而实现外泌体内miRNA的原位、快速、准确检测。该方法无需预先分离破坏外泌体，可避免miRNA的降解，检测结果更准确。此外病毒融合蛋白介导的囊泡与外泌体融合，具有融合效率高、检测速度快的优点。本项目为检测外泌体内miRNA提供新的方法，对肿瘤的早期诊断、疗效评估和预后分析具有重要意义。

外泌体作为一种内源性的细胞外囊泡，具有免疫原性低、无毒性和长血液循环时间等优势。外泌体内miRNA可以作为临床肿瘤诊断和治疗疗效检测的重要标志物。近年来检测外泌体内miRNA的方法层出不穷，但多存在耗时、回收率低和成本昂贵的问题。此外,目前检测外泌体miRNA的主要方法在提取miRNA时还需破坏外泌体，容易造成miRNA的损失并影响结果准确性。因此亟需建立一种可以简单、快速、准确检测外泌体内miRNA的方法。受病毒融合细胞膜入侵细胞的启发，本项目构建了一种仿病毒融合囊泡，通过囊泡表面的血球凝集素-神经氨酸酶蛋白结合外泌体表面唾液酸受体，随后融合蛋白经历构象变化插入外泌体膜，引起囊泡与外泌体的高效融合，从而将囊泡内的分子信标递送进入外泌体。与外泌体内特异miRNA结合后，分子信标荧光信号开启，从而实现外泌体内miRNA的原位、快速、准确检测。研究结果显示，仿病毒融合囊泡借助流式细胞术不仅可以检测外泌体miRNA，还可以识别肿瘤相关miRNA来区分肿瘤外泌体和正常外泌体。综上，该方法无需预先分离破坏外泌体，可避免miRNA的降解，检测结果更准确。此外病毒融合蛋白介导的囊泡与外泌体融合，能够在2小时内快速，高效和高通量地检测外泌体miRNA。本项目为快速高效地检测外泌体内miRNA提供新的方法，对肿瘤的早期诊断、疗效评估和预后分析具有重要意义。