重组病毒在循环肿瘤细胞检测中的应用及研究进展

Detection of circulating tumor cells by recombinant virus

循环肿瘤细胞（Circulating tumor cells,CTCs）是液体活检的重要手段之一,也是非侵入性的个体化实验诊断工具.以FDA首个批准的CTCs检测平台CellSearch为代表的商品化CTCs分离平台已被证实可有效富集监测CTCs,但出现上皮—间质转化的CTCs因不表达上皮细胞粘附分子而可能被漏检.此外大多CTCs检测平台因无法区分活细胞和死细胞而不能有效反映CTCs功能状态,因此如何利用常规设备开发新型分离方法用于检测具有转移潜力的CTCs具有更加重要的现实意义.插入人端粒酶启动子和绿色荧光蛋白（Green fluorescent protein,GFP）的复制选择性溶瘤病毒,主要为单纯疱疹病毒（Herpes simplex virus,HSV）和腺病毒（Adenovirus,ADV）具有特异示踪CTCs的作用.肿瘤细胞感染重组病毒24 h后稳定表达GFP,通过荧光显微镜或流式细胞仪直接检测荧光信号即可对CTCs进行计数,结合PCR、测序等方法亦可进行相关下游分析.此外,通过基因工程改造的复制选择性溶瘤病毒仅在活细胞中生长繁殖,有利于在早期发现具有转移潜能的高活性CTCs.以重组病毒为基础的CTCs检测方法灵敏准确、简便易行,具有较好的临床应用前景.

The detection and molecular characterization of circulating tumor cells （CTCs） is one of the most important tool for liquid biopsy,which has the potential to enable non-invasive diagnostic tests for personalized medicine.Commercial platforms represented by CellSearch,the first FDA approved assay,have been considered to be valid for CTCs detection.However,special equipment and consumptive materials are required in the techniques listed above.Besides,most of them can not differentiate between apoptotic and viable cells,which indicates the portion of active and functional CTCs.Therefore,how to develop novel nethod for CTCs enrichment with metastatic potential has great significance in clinical routine.Telomerasespecific replication-selective oncolytic viruses expressing green fluorescent protein （GFP）,including herpes simplex virus and adenovirus,allow the detection for human CTCs in the peripheral blood.After 24 h of transfection with recombinant virus,the tumor cells stably express GFP,and it could be used for CTCs counting by fluorescent microscopy or flow cytometry.Moreover,downstream analysis would be achieved by combination with PCR or DNA sequencing.Recombinant virus enables early detection of metastatic tumor cells,because the fluorescent signal is amplified only in viable,infected CTCs,by viral replication.This GFP-expressing virus-based method is remarkably sensitive,simple,and feasible,and it offers a new opportunity to detect and characterize CTCs in clinical routine.