表达绿色荧光蛋白的单纯疱疹病毒检测循环肿瘤细胞  

Identification of occult disseminated tumor cells by recombinant herpes simplex virus expressing GFP （ HSV^GFP ）

作　　者：韩香萍[1] 施桂兰[2] 王成锋[3] 李洁[2] 张建伟[3] 张郁[2] 张叔人[2] 刘滨磊[2] 

机构地区：[1]南开大学生命科学学院,天津300071 [2]中国医学科学院北京协和医学院肿瘤医院肿瘤研究所免疫室 [3]中国医学科学院北京协和医学院肿瘤医院肿瘤研究所免疫室腹部外科

出　　处：《中华肿瘤杂志》2012年第12期894-898,共5页Chinese Journal of Oncology

基　　金：天津市科委科技支撑重点项目（10ZCKFSY08000）;中国医学科学院肿瘤医院重大课题（LC20IOZ01）

摘　　要：目的以表达绿色荧光蛋白（GFP）的单纯疱疹病毒（HSV^GFP）建立一种快速检测循环肿瘤细胞（CTCs）的方法，并与常规细胞学检测方法的检测结果进行比较。方法将HSV^GFP与7个不同的肿瘤细胞系共培养，用荧光显微镜观察表达荧光的细胞。将10、100、1000和10000个人肺癌PG细胞加入到2ml全血中，以淋巴细胞分离液清除红细胞，同时富集肿瘤细胞。以HSV^GFP感染收集到的细胞，用荧光显微镜确定荧光细胞数量，分析HSV^GFP检测方法的敏感性。以HSV^CFP法检测70例肿瘤患者临床样本中的CSCs，并与其中15例有细胞学报告者的检测结果进行比较。结果HSV^GFP感染的7个肿瘤细胞系均表达绿色荧光。应用HSV^GFP检测方法，可以在仅加入了10个肿瘤细胞的2ml全血中检测出阳性结果。对70例临床样本采用HSV^GFP法进行检测的结果显示，有40例样本呈阳性反应。在15例有细胞学检测结果的临床样本中，细胞学检测阳性仅有4例，而HSV^GFP方法检测阳性有11例；其中细胞学检测阳性的4例样本，HSV^GFP检测均为阳性。结论以HSV^GFP检测患者体内CTCs的方法简便，且具有较高的敏感性。Objective To develop a novel rapid protocol for the detection of occult disseminated tumor cells by a recombinant herpes simplex virus expressing GFP （ HSV^GFP ）. Methods Tumor cells of seven cell lines were exposed to HSV^GFP and then examined for GFP expression by fluorescence microscopy. Various numbers of tumor cells （10, 100, 1000, 10 000） were mixed into 2 ml human whole blood, separated with lymphoeytes separation medium, exposed to HSV^GFP, incubated at 37℃ for 6-24 h and then counted for the number of green cells under the fluorescence microscope. Some clinical samples including peripheral blood, pleural effusion, ascites, spinal fluid from tumor-bearing patients were screened using this protocol in parallel with routine cytological examination. Results HSV^GFP was able to infect all 7 tumor cell lines indicating that the HSV^GFP can be used to detect different types of tumor cells. The detection sensitivity was 10 cancer cells in 2 ml whole blood. In the clinical samples, there were 4/15 positive by routine cytological examination but 11/15 positive by HSV^GFP, indicating a higher sensitivity of this new protocol. Condusion Recombinant herpes simplex virus-mediated green fluorescence is a simple and sensitive technique for the identification of occult disseminated cancer cells including circulating tumor cells （CTCs）.

关 键 词：单纯疱疹病毒属 绿色荧光蛋白质类 诊断试验 常规 循环肿瘤细胞 

分 类 号：R73[医药卫生—肿瘤]