纳米免疫磁珠联合巢式PCR检测胰腺癌患者外周循环微转移的意义

Combination of immunomagnetic nanobeads and RT-nest-PCR for detection of micrometastases of pancreatic cancer in peripheral circulation

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 develop an effective method to detect circulating micrometastases in patients with pancreatic cancer. Methods： Peripheral blood mononuclear cells（PBMC） from normal subject were mixed with human pancreatic cancer PANC-1 cells in different proportions.The mixed cells of each proportion were sorted by using EpCAM-antibody-coated immunomagnetic nanobeads for separation and enrichment of the tumor cells.Then,the expression of human telomerase reverse transcriptase（h-TERT） and c-met in the tumor cells were detected by RT-nest-PCR,so as to determine the sensitivity of this method.The expressions of h-TERT and c-met in the PBMC from 25 patients with pancreatic cancer and 15 patients with benign diseases were detected by using the above combined immunomagnetic nanobeads and RT-nest-PCR method.The relations of both genes with the clinicopathologic factors were also analyzed. Results： The sensitivity of the combined immunomagnetic nanobeads and RT-nest-PCR method for h-TERT and c-met was 1 tumor cell/1×10^7 PBMC and 1 tumor cell/1×10^6 PBMC,respectively.The expression rates of h-TERT and c-met both were 0（0/15） in benign patients,and was 100%（25/25） and 80%（20/25） in pancreatic cancer patients,respectively.Moreover,the positive expression rate of c-met was significantly related to the tumor stage（P〈0.05）. Conclusion： Detection of h-TERT and c-met using combined immunomagnetic nanobeads and RT-nest-PCR is a highly sensitive method for identifying the micrometastases of pancreatic cancer in peripheral circulation.