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目的建立反转录结合荧光定量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,制备标准品并做测序验证。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,反转录结合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103拷贝/微升标准品重复检测5次,x±s:718.9±120.5,CV值:16.7%;MDA-MB453细胞的血标本做灵敏度试验,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 establish a reverse transcription combined with fluorescence quantitative PCR (FQ-PCR) detection of hMAM gene expression in peripheral blood of breast cancer cells to understand its value of monitoring micrometastasis in breast cancer cells. Methods Clone the target fragment, prepare standards and do sequencing verification. The breast cancer MDA-MB453 and MCF7 cell lines were cultured to make the blood sample model. The GAPDH gene was used as the internal control. Reverse transcription and FQ-PCR were used to detect the expression of hMAM in breast cancer cell lines and 10 cases of breast cancer. Results Electrophoresis and sequencing confirmed that the cloned product was the desired fragment of interest. The correlation coefficient of the FQ-PCR standard curve was -1.0; the detection was repeated 5 times with 103 copies / microliter standard, x ± s: 718.9 ± 120.5, CV value: 16.7%; the sensitivity of the blood sample of MDA-MB453 cells was tested, Equivalent to 104 blood cells containing 1 breast cancer cells can be detected positive. No hMAM expression was detected in MCF7 cells. Of the 10 breast cancer patients, among 5 cases of lymph node metastasis, 3 were hMAM positive and 2 were negative. Among the 5 cases without lymph node metastasis, 1 showed hMAM expression positive and 4 were negative. Conclusion This method can effectively monitor the micrometastasis of breast cancer cells in peripheral blood and found that the expression of hMAM in different breast cancer cell lines varies greatly.