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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。
The purpose of the circulating tumor cells in the diagnosis of tumor metastasis, individualized treatment / efficacy evaluation and tumor metastasis mechanism research has great value, however, due to its very low proportion in the circulatory system, the separation and identification of circulating tumor cells become clinical difficulties. oHSV1-GFP is a genetically modified human herpes simplex virus. We replaced the promoter of ICP4, a key gene for replication of the virus, with the promoter of human telomerase reverse transcriptase and ligated the downstream of ICP4 with the GFP expression cassette so that after infection of the virus, the virus can be expressed in the telomerase reverse transcriptase Transcriptional Environment Activates Downstream Green Fluorescent Protein Gene Expression. The purpose of this study is to use the virus to capture circulating tumor cells, so as to establish a simple and reproducible detection method with high sensitivity and to explore the reliability and clinical value of the detection method. Methods The expression of telomerase reverse transcriptase in tumor cell lines, tumor tissues and normal cells was detected by Western blot. 4 ml of peripheral blood from normal healthy volunteers was collected and the tumor cell lines were added to peripheral blood according to a certain gradient. According to MOI = 1 The percentage of CD45- / GFP + cells was detected by flow cytometry, and the detection rate of this method was determined. 4ml of peripheral blood from clinical specimens was collected, and the erythrocytes were lysed and removed, and then transfected at a MOI = 1 ratio Recombinant virus oHSV1-GFP DNA was collected 24 h later. CD45- / GFP + cells were collected and counted by flow cytometry. The clinical application of this method was preliminarily tested. Results Western blot results showed that the tumor cell lines and tumor tissues showed high expression of telomerase reverse transcriptase, while the expression of telomerase reverse transcriptase was low in normal cells. In this assay, we define CD45- / GFP + cells as circulating tumor cells. The detection rate of SMMC7721 cells in peripheral blood samples showed that the numbers of 10, 50 and 100 (detection) of peripheral blood were 6.8 ± 2.04, 40.8 ± 4.04 and 85.9 ± 7.03, respectively. The overall detection rate was 87.9%. Huh7 The detection numbers of 10, 50 and 100 cells were 6.6 ± 1.90, 39.8 ± 3.88 and 86.4 ± 7.63, respectively. The overall detection rate was 88.5%. Linear regression analysis showed that the number of cells captured by the method and the actual number of tumor cells showed a significant correlation, r2> 0.95. Peripheral blood samples from 26 patients with HCC and 50 healthy controls were detected by this method. The number of CD45- / GFP + cells (16.7 ± 2.77) in patients with HCC was significantly higher than that in healthy controls (0.94 ± 0.12, P <0.0001) Subgroup analysis showed that the number of circulating tumor cells in peripheral lymph nodes (23.77 ± 4.49) was significantly higher in patients with regional lymph nodes than in regional lymph nodes (9.76 ± 1.87), P = 0.0002. Conclusion This study established a new detection method of circulating tumor cells, and evaluated the reliability and clinical value of this method. The method can specifically track circulating tumor cells and has potential clinical application value.