实时荧光定量RT-PCR检测急性白血病中Bmi-1基因mRNA的表达及意义

Expression and significance of Bmi-1 mRNA in acute myeloid leukemia with real-time fluorescent quantitative polymerase chain reaction

[目的]建立荧光实时定量RT—PCR（FQRT—PCR）检测Bmi—1基因mRNA的方法并研究该基因在急性白血病细胞中表达的意义。[方法]根据Genbank提供的序列，设计目的基因Bmi-1及内参基因GAPDH的扩增引物，将Bmi-1及GAPDHRT—PCR扩增片段克隆入载体pMDl8-Tsimple，经测序鉴定正确后，进行纯化、定量及系列稀释，应用SYBRGreenI荧光染料，建立检测Bmi-1mRNA的FQRT—PCR方法，并对其线性及特异性进行评价。应用此方法检测21例急性白血病及10例健康人外周抗凝血中Bmi-1mRNA的表达水平。【结果]该方法的线性范围为1．0×10^3-1．0×10^8eopies／txL，相关系数为-1．00，熔解曲线分析扩增产物显示单-的峰，熔解温度（Tm）为80．4℃。急性白血病患者的相对表达量为0．56±0．12，健康对照组中Bmi-1mRNA的相对表达量为0．19±0．08，两者的表达差异有统计学意义（P〈0．05）。[结论]实时荧光定量RT—PCR方法检测Bmi-1基因表达，具有高敏感性和高特异性等优点，可作为进-步研究Bmi-1基因的方法。Bmi-1基因参与急性白血病的发生，可能是一种新的肿瘤标志物。

[ Objective] To establish a real -time fluorescent quantitative polymerase chain reaction method for quantifying Bmi - 1 mRNA and study its expression and significance in acute myeloid leukemia. [ Methods] The specific primers of Bmi - 1 and GAPDH were designed according to GenBank database. The Bmi - 1 and GAPDH fragments in pure form from classical RT- PCR were cloned to pMD18 -T simple vector respectively, and reconflainant plasmids were purified and quanti- fied speetrophotometrically. Using SYBR Green I, a real - time fluorescent quantitative PCR method was established, and its linearity and specificity were evaluated. This method was employed to detect the expression of Bmi - 1 mRNA in acute myeloid leukemia and normal controls. [ Results] The linear range was 1.0 ×10^3- 1.0 ×10^8copies/μL. The relation coefficient was - 1.00. Melting curve analysis showed a single peak, and Tm was 80.4℃. The relative expression level of Bmi - 1 mRNA in acute myeloid leukemia and normal controls was 0.19 ± 0.08 and 0.56 ± 0.12, respectively （ P 〈 0.05 ）. [ Conclusions ] SYBR Green I real - time fluorescent quantitative polymerase chain reaction which is specific, sensitive and accurate can be practiced to further research on Bmi - 1 gene. Bmi - 1 may contribute to leukemogenesis and may be a new tumormarker.