实时荧光PCR检测癌基因MDM_2在急性白血病中的表达

Detection of MDM_2 Oncogene Expression in Acute Leukemia by Real-Time Fluorescence PCR Assay

目的 应用实时荧光PCR技术 ,检测癌基因MDM2 在急性粒细胞白血病 (AML)中的表达并分析其与AML发生、分型及疗效的关系。方法 以SybrGreenⅠ为荧光指示剂 ,β actin作内参照 ,建立检测MDM2 mRNA表达的实时荧光PCR反应体系。PCR反应完毕作熔解曲线分析。分析MDM2 mRNA的表达与AML的发病、FAB分型及疗效的关系。结果 ①通过对实验参数的优化 ,适宜的反应条件为退火温度 5 1℃ ,Mg2 + 浓度 3mmol/L ,牛血清白蛋白 (BSA)浓度 0 1mg/ml,循环次数 4 5次。批内、批间变异系数分别为 8 81%和 15 39%。②AML组MDM2 mRNA的表达明显高于正常对照组 (P <0 0 1) ;AML各亚型M1、M2 与M3 组MDM2 mRNA表达量组间差异无显著性意义(P >0 0 5 ) ;完全缓解组、部分缓解组、未缓解组 3组之间MDM2 mRNA表达差异有显著性意义 (P <0 0 1) ,且呈逐渐上升趋势。结论 采用SybrGreenⅠ的Lightcycler实时荧光PCR方法检测MDM2 mRNA快速、简便、稳定性好。MDM2 mRNA的表达与AML的发生及临床疗效有重要关系。

Objective To apply a real-time fluroscent PCR assay for detecting MDM 2 mRNA expression in acute granulocytic leukemia (AML) and study the correlation of MDM 2 mRNA with pathogenesis, FAB classification and treatment outcome. Methods Using the fluorescent indicator Sybr Green Ⅰ and an inner control β-actin gene, an amplifying condition was established for detecting MDM 2 gene by Lightcycler instrument. After PCR melting curve analysis was done. The correlations of MDM 2 mRNA with pathogenesis, FAB classification and treatment outcome were studied. Results By optimizing the parameters, the suitable reaction conditions were that the annealing temperature was 51 ℃, the concentration of magnesium was 3 mmol/L, the concentration of BSA was 0.1 mg/ml and the cycle times was 45. The intro group CV and inter group CV were 8.81 % and 15.39 % respectively. The expression of MDM 2 mRNA in AML group was markedly higher than in normal control group (P<0.01). There was no significant difference in the MDM 2 mRNA expression among the FAB types (P>0.05). There was significant difference in the MDM 2 mRNA expression among the complete remission group (CR), partial remission group (PR) and no response group (NR) (P<0.01). Conclusion The assay was rapid, easy and stable. The expression of MDM 2 mRNA was closely correlated with the pathogenesis of AML and treatment outcome.