免疫荧光动态监测急性早幼粒细胞白血病患者PML-RARα融合蛋白变化

摘要

背景:实时定量RT-PCR 只能间接地反映融合基因表达量的变化,并不能在细胞原位直观反映急性早幼粒细胞白血病患者细胞内PML-RARα融合蛋白在全反式维甲酸治疗前后的相对表达量.目的:探讨免疫荧光技术检测融合蛋白PML-RARα表达的动态变化在判断急性早幼粒细胞白血病的全反式维甲酸早期治疗的效果.方法:应用免疫荧光技术检测全反式维甲酸干预的急性早幼粒细胞白血病经典细胞株NB4 细胞或来源于急性早幼粒细胞白血病患者的NB4 细胞PML-RARα蛋白的表达,以实时定量RT-PCR 检测细胞PML-RARα mRNA 的表达作为对照,以评价免疫荧光技术的可靠性.结果与结论:免疫荧光染色显示,无论是体外培养的NB4 细胞,还是患者骨髓内的NB4 细胞,在全反式维甲酸干预前细胞内均可见大量红色的融合蛋白PML-RARα,大小不均一、形状不规则,全反式维甲酸干预后,NB4 细胞内红色的融合蛋白PML- RARα形状逐渐规则,弥散状态变为散在分布,且随着全反式维甲酸干预时间的延长,原有的弥散状态逐渐消失.实时定量RT-PCR 测得的PML-RARα mRNA 的变化趋势与免疫荧光相一致.说明免疫荧光技术检测PML-RARα能够更为直观地判断全反式维甲酸的治疗效果.%BACKGROUND: Real-time quantitative RT-PCR can directly reflect the expression change of fusion gene, but it cannot directly reflect the relative expression of intracellular PML-RARα fusion protein in patients with acute promyelocytic leukemia (APL) before and after all-trans retinoic acid treatment. OBJECTIVE: To investigate the reliability of immunofluorescence technique in judging the early-stage therapeutic effects of all-trans retinoic acid treatment in APL by dynamic monitoring PML-RARα fusion protein. METHODS: Immunofluorescence technique was used for detection of PML-RARα protein expression in typical APL cell strain BN4 cells or NB4 cells of patients with APL following all-trans retinoic acid treatment. Real-time quantitative RT-PCR was used for detection of intracellular PML-RARα mRNA expression as control to investigate the reliability of immunofluorescence technique.RESULTS AND CONCLUSION: Immunofluorescence staining results showed that before all-trans retinoic acid treatment, a large amount of red fusion protein PML-RARα of different size and irregular morphology was observed in BN4 cells in vi tro cultured or from patients' bone marrow; after all-trans retinoic acid treatment, PML-RARα fusion protein in the NB4 cells gradually became regular and was gradually scattered with prolongation of treatment time. Real-time quantitative RT-PCR results showed that the change tendency of PML-RARα mRNA expression was in accordance with immunofluorescence results. This suggests that immunofluorescence for detection of PML-RARα expression can be used for directly judging the therapeutic effects of all-trans retinoic acid treatment.