建立人乳腺珠蛋白mRNA FQ-PCR方法用于乳腺癌微小转移的诊断

Establishment 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(hum an m amm aglob in,hM aM)基因,建立荧光定量PCR方法,探讨外周血hM aM 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hM aM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hM aM 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),hM aM mRNA的表达与乳腺癌病理分期无关(χ2=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hM aM mRNA。结论hM aM 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 （FQ-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. A FQ-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 （ X^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 （ X^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 mierometastasis of breast cancer through blood.