【摘要】 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。更多还原

【Abstract】 Objective To clone human manunaglobin (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 mierometastasis 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 stageⅠ,3 of stageⅡ,1 of stageⅢand 1 of stageⅣ).There was no relationship between hMaM mRNA expression arid 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 micrometastasis of breast cancer through blood.更多还原