外周血中检测乳腺癌细胞株hMAM表达的方法探讨

The evaluation of the method detecting hMAM gene expression of breast cancer cell lines in peripheral blood

目的建立逆转录结合荧光定量PCR(FQ-PCR)检测外周血乳腺癌细胞hMAM基因表达方法,了解其监测乳腺癌细胞微转移的应用价值。方法克隆目的片段,制备标准品并作测序验证。培养乳腺癌MDA-MB453、MCF7细胞株,制成血液标本模型,GAPDH基因为内对照,逆转录结合FQ-PCR检测乳腺癌细胞株血标本模型和10例乳腺癌患者外周血的hMAM表达。结果电泳和测序证实,克隆产物系预期的目的片段。FQ-PCR标准曲线的相关系数为-1.0;用103copies/μl标准品重复检测5次,x±s:718.9±120.5,CV16.7%;MDA-MB453细胞的血标本作灵敏度试验,相当于104个血细胞中含1个乳腺癌细胞可检出阳性。未检测到MCF7细胞的hMAM表达。10例乳腺癌患者,淋巴结转移的5例中,3例hMAM表达阳性、2例阴性;无淋巴结转移的5例中,1例hMAM表达阳性、4例阴性。结论本方法可有效监测外周血中微转移的乳腺癌细胞,发现不同乳腺癌细胞系hMAM表达程度差异大。

Objective To evaluate the method detecting hMAM gene expression of breast cancer cell lines in peripheral blood.Methods The hMAM gene was cloned and confirmed by agarose gel electrophoresis and sequence analysis,the standard substance of real-time fruorescence quantitative polymerase chain reaction (FQ-PCR )was made. The breast cancer cell lines MDA-MB453、MCF7 were cultured and the blood samples model was made.The hMAM expression of breast cancer cell lines blood model and 10 cases of breast cancer patient peripheral blood were detected by RT-PCR and FQ-PCR,GAPDH gene expression as contrast. Results The production of PCR was cloned and testified by sequence.The relative coefficient(r) of standard curve of FQ-PCR was -1.0. The x±s and CV were 718.9±120.5 and 16.7%, respectively. The hMAM expression of 1 breast cancer cell lines MDA-MB453 in 104 blood cells could be detected, but that of breast cancer cell line MCF7 was not be detected. The hMAM expression was detected in 3 of 5 patients with sentinel nodes and in 1 of 5 patients with non-sentinel nodes.Conclusion It might be a good method for detecting micrometastased breast cancer cell in peripheral blood. The diffrernce of hMAM gene expression correlates with the type of breast cancer cell lines.