荧光定量PCR法检测人乳腺珠蛋白与乳腺癌微小转移诊断的研究

The Study of FQ-PCR for Determining Mammaglobin mRNA and Implication for Monitoring Micrometastasis of Patients with Breast Cancer

目的克隆人乳腺珠蛋白（hMaM）基因,建立荧光定量PCR方法,探讨外周血hMaM mRNA的表达作为乳腺癌血行微小转移标志物的可能性。方法设计特异引物,用RT-PCR方法从乳腺癌组织扩增获得目的片段,利用T/A克隆将PCR产物插入pMD-18T载体,优化扩增条件,以pMD/M质粒制备标准曲线,建立FQ-PCR方法,对乳腺癌不同分期的患者（n=28）,乳腺良性疾病患者（n=15）,其他肿瘤患者（n=33）的外周血进行检测。结果成功获得hMaM基因并完成克隆,阳性克隆测序结果与预期序列一致;建立了FQ-PCR检测hMaM mRNA方法;28例乳腺癌病人中9例（32.1%）阳性（Ⅰ期4例,Ⅱ期3例,Ⅲ期1例,Ⅳ期1例）,hMaM mRNA的表达与乳腺癌病理分期无关（χ^2=0.5936;P〉0.05）;乳腺良性疾病组,其他肿瘤病人组及对照组外周血中均未检出hMaM mRNA。结论hMaM mRNA是乳腺癌特异性标志物,其外周血的表达可作为乳腺癌血行微小转移的指标。

Objective To clone human mammaglobin （hMaM） gene and establish a method of fluorescence quantitative PCR for determining hMaM mRNA, and investigate the possibility of hMAM mRNA as a biomarker for monitoring micrometastasis of breast cancer in peripheral blood. Methods The interested segment was obtained by reverse transcription PCR with the designed specific primers, and inserted into pMD-18T vector by T/A match. FQ-PCR was established after optimizing the system of PCR amplification and the standard curve was generated by series dilution of pMD/M plasmid DNA templates, hMaM mRNA of peripheral blood monocytes was isolated from patients,including breast cancer group （n=28）, benign breast diseases group （n=15） and other cancers group （n =33）,and healthy persons （n =18） and was determined by FQ-PCR. Results hMaM gene was obtained and inserted into pMD-18T vector. Sequencing result of the positive clone was accordant with the expected sequence. AFQ-PCR for detection of hMAM mRNA was established successfully, hMAM mRN was positive in 9 out of 28（32.1%）blood sample of the patients with breast cancer （4 of stage Ⅰ,3 of stage Ⅱ, 1 of stage Ⅲ and 1 of stage Ⅳ ）. There was no relationship between hMaM mRNA expression and pathological stages （χ^2 = 0.5936, P 〉 0.05 ）. There were obvious differences between breast cancer group and benign breast diseases group,other cancers group or healthy persons group in the expression of hMaM mRNA （ χ^2 = 8.96,13.49 and 10.32 respectively,P 〈 0.005）. Conclusion hMaM mRNA is a characteristic biomarker of breast cancer and its expression in peripheral blood indicates micrometastasis of breast cancer through blood.