实时定量逆转录PCR检测急性髓系白血病患者骨髓细胞BAALC基因表达

Detection of BAALC expression in bone marrow of acute myeloid leukemia patients by real-time quantitative reverse transcriptase polymerase chain reaction

目的:建立实时定量逆转录聚合酶链反应(RQ-PCR)检测急性髓系白血病(AML)骨髓细胞BAALCmRNA的方法,并对24例初诊AML患者骨髓标本进行检测,初步观察其表达及意义。方法:构建BAALC基因转录本的阳性标准品质粒,利用ABI Prism7300型实时定量PCR仪进行检测,BAALC mRNA定量结果以校正比值(NQ)表示,NQ=BAALC mRNA拷贝数/ABL mRNA拷贝数。结果:RQ-PCR实验敏感度为5个拷贝。ABL及BAALC基因CT值的批内差及批间差均<2.26%。FAB各亚型中,M2型BAALC基因高表达率高于M3-M6型(P<0.05)。BAALC高表达患者血小板计数倾向于比低表达患者更低(P=0.175),CD34阳性率倾向于比低表达患者高(P=0.153)。骨髓增生异常综合征(MDS)转化的或具有MDS特点的AML(MDS-related AML)患者无论BAALC高表达还是低表达均呈现难治/复发,而原发性AML患者BAALC高表达者难治/复发率倾向于比低表达者高(P=0.182)。结论:建立的RQ-PCR检测AML患者骨髓细胞BAALC mRNA的方法稳定可靠,敏感度高。BAALC基因高表达可能是原发性AML患者一个重要不良预后因素。

Objective：To establish a real-time quantitative reverse transcriptase polymerase chain reaction（RQ-PCR） assay for detecting the expression of BAALC （brain and acute leukemia,cytoplasmic）mRNA.The bone marrow samples from 24 newly diagnosed acute myeloid leukemia （AML）patients were collected for anaiysis. Methods：A plasmids containing cDNA fragments of BAALC gene were constructed.The ABI Prism7300 Sequence Detection System using Taqman fluorogenic probes was used to quantify target gene.BAALC mRNA was detected by RQ-PCR in 24 AML patients.The normalized quotient （NQ）of BAALC mRNA was calculated as followings：NQ=BAALC mRNA copy numbers/ABL mRNA copy numbers.Results：The sensitivity of RQ-PCR was 5 copies.The coefficients CT of interassay and intraassay variation for ABL and BAALC were all below 2.26%.High BAALC expression was more predominantly seen in FAB M2 than in FAB M3-M6 （P〈0.05）. Compared with low expressers,high BAALC expressers tended to have lower platelet counts （P=0.175）and a higher positive rate of CD34（P=0.153）.All myelodysplastic syndromes（MDS）-related AML patients with high or low BAALC expression predicted refractoriness or impending relapse.De novo AML patients with high BAALC expression tended to relapse or be refractory more frequently than those with low BAALC expression （P= 0.182）.Conclusion：A stable,reliable and sensitive RQ-PCR method was established to detect the expression of BAALC mRNA in bone marrow of AML patients.High BAALC expression predicts an adverse prognosis and may define an important risk factor in de novo AML.