oHSV1-GFP法检测肝癌循环肿瘤细胞

A novel detection method for circulating tumor cells

目的循环肿瘤细胞在肿瘤转移诊断、个体化治疗/疗效评估及肿瘤转移机制研究等方面具有重要价值,然而由于其在循环系统中比例极低,循环肿瘤细胞的分离和鉴定成为临床应用的难点。oHSV1-GFP是一种经过基因修饰的人单纯疱疹病毒。我们将该病毒的复制关键基因ICP4的启动子替换为人端粒酶逆转录酶的启动子,并在ICP4下游连接GFP表达盒,以使该病毒在感染细胞后,能在端粒酶逆转录酶转录环境的作用下激活下游绿色荧光蛋白的表达基因。本研究旨在利用该病毒捕获循环肿瘤细胞,从而建立一种简便重复性好灵敏度高的检测方法,并对该检测方法的可靠性及临床应用价值进行探讨。方法 Western blot技术检测肿瘤细胞系、肿瘤组织及正常细胞中端粒酶逆转录酶表达情况;抽取正常健康人外周血4ml,将肿瘤细胞系分别按一定梯度加到外周血中,按照MOI=1比例转染重组病毒oHSV1-GFP DNA,流式细胞仪检测CD45-/GFP+细胞数,进行该方法检出率的测定;收集临床标本外周血4ml,裂解去除红细胞后,按照MOI=1比例转染重组病毒oHSV1-GFP DNA,24h后收集细胞,流式细胞仪检测CD45-/GFP+细胞并计数,初步检测该方法临床应用的可行性。结果 Western blot检测结果显示肿瘤细胞系及肿瘤组织呈现端粒酶逆转录酶高表达状态,而正常细胞中端粒酶逆转录酶表达量低。在该检测方法中,我们将CD45-/GFP+细胞定义为循环肿瘤细胞。检出率测定结果显示:外周血中加入SMMC7721细胞10,50,100(个)的检出数分别为6.8±2.04,40.8±4.04,85.9±7.03,总体检出率为87.9%,外周血中加入Huh7细胞10,50,100(个)的检出数分别为6.6±1.90,39.8±3.88,86.4±7.63,总体检出率为88.5%。线性回归分析显示该方法捕获的细胞数与实际肿瘤细胞数呈明显相关性,r2>0.95。应用该方法检测26例肝癌患者及50例正常健康人外周血显示,肝癌患者外周血中CD45-/GFP+的细胞数(16.7±2.77)明显高于正常健康人(0.94±0.12)P<0.0001,亚组分析显示区域淋巴结阳性的患者外周血中循环肿瘤细胞计数(23.77±4.49)明显高于区域淋巴结(9.76±1.87)的患者,P=0.0002。结论本研究建立了一种新型循环肿瘤细胞检测方法,并对该方法的可靠性及临床应用价值进行了评估。该方法能够特异性示踪循环肿瘤细胞,具有潜在临床应用价值。

Objective Blood tests of circulating tumor cells have shown great potential to early diagnose tumor metastasis,carry out individualized treatment,evaluate treatment efficacy and investigate the mechanism of tumor metastasis.It is difficult to identify the CTCs as they are an extremely rare phenomenon.Our research group constructed oHSV1-GFP.The virus could proliferate specifically in cells which have telomerase activity and then express GFP.This study aims to utilize the virus to establish a simple,reproducible,and high sensitivity detection approach.The reliability of the test method and the value of clinical application were discussed.Methods The hTERT expression levels of different tumor cell lines,tissues and normal cells were detected by Western blot assay.Tumor cell lines were used to simulate the tumor patients with peripheral blood to determine recovery of this method.At last the clinical samples,as little as 4ml peripheral blood,were used to detect CTCs by this method.Results The hTERT was highly expressed in tumor cell lines and tumor tissues.The CD45-/GFP+cells were defined as CTCs by this approach.The recovery rates of SMMC7721 and Huh7were >85%.Regression analysis of the number of CD45-/GFP+ cells versus the number of spiked tumor cells showed good relevancy.CTCs in 25 hepatoma samples and 50 healthy samples were evaluated and the number of CD45-/GFP+ cells in hepatocellular carcinoma patients' peripheral blood(23.77±4.49)was significantly higher than in healthy people(0.94±0.12)(P<0.0001).The number of CD45-/GFP+cells in regional lymph node positive patients' peripheral blood(23.77±4.49)was significantly higher than in regional lymph node negative patients(9.76±1.87)(P=0.0002.)Conclusion We conclude that this clinical applicable novel technique is a simple and repeatable CTC detection method and has potential clinical application value.