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目的:建立一种检测胰腺癌患者外周血循环微转移的有效方法。方法:用人胰腺癌细胞株PANC-1与正常人外周血单核细胞(PBMC)以不同比例混合,用EpCAM抗体的纳米免疫磁珠对各比例组进行阳性分选,分离富集出肿瘤细胞,用巢式RT-PCR(RT-nest-PCR)检测细胞中端粒酶亚催化单位(h-TERT)和c-met的表达,以确定该检测方法的敏感度;用以上纳米免疫磁珠联合RT-nest-PCR方法检测25例胰腺癌患和15例良性病患者PBMC中h-TERT和c-met的表达,并分析两者与胰腺癌患者临床病理因素的关系。结果:纳米免疫磁珠联合RT-nest-PCR法检测h-TERT和c-met的敏感度分别为1个肿瘤细胞/1×107个PBMC和1个肿瘤细胞/1×106个PBMC。良性疾病组h-TERT及c-met基因表达率均为0(0/15),胰腺癌组h-TERT和c-met基因表达率分别为100%(25/25)和80%(20/25),且c-met阳性表达率与肿瘤分期有关(P<0.05)。结论:免疫磁珠联合RT-nest-PCR法检测h-TERT和c-met是发现胰腺癌外周血循环微转移的高敏感度方法。
Objective: To establish an effective method for detecting micrometastasis in peripheral blood of patients with pancreatic cancer. Methods: Human pancreatic cancer cell line PANC-1 and normal human peripheral blood mononuclear cells (PBMC) were mixed in different proportions. EpCAM antibody nano-immunomagnetic beads were used for positive sorting of each proportion group, and the tumor cells were isolated and enriched. The expression of telomerase subunit (h-TERT) and c-met in the cells was detected by nested RT-PCR (RT-nest-PCR) to determine the sensitivity of the detection method. RT-nest-PCR was used to detect the expression of h-TERT and c-met in PBMC from 25 patients with pancreatic cancer and 15 patients with benign disease. The relationship between h-TERT and c-met expression and the clinicopathological factors were analyzed. Results: The sensitivity of nano-immunomagnetic beads combined with RT-nest-PCR in detecting h-TERT and c-met was 1 tumor cell / 1 × 107 PBMC and 1 tumor cell / 1 × 106 PBMC, respectively. The expression rates of h-TERT and c-met in benign disease group were all 0 (0/15). The expression rates of h-TERT and c-met in pancreatic cancer group were 100% (25/25) and 80% (20 / 25), and the positive rate of c-met correlated with tumor staging (P <0.05). Conclusion: The combination of immunomagnetic beads and RT-nest-PCR to detect h-TERT and c-met is a highly sensitive method for detecting micrometastasis in the peripheral blood of patients with pancreatic cancer.