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目的探讨全反式维甲酸(ATRA)对K562白血病细胞中铁代谢相关基因表达产物(H-Fn、TfR及IRP2)的影响及其相互关系。方法应用联苯胺、瑞氏、非特异性酯酶、硝基四氮唑蓝还原试验4种染色方法 ,观察ATRA对K562细胞的诱导分化特点;应用流式细胞术检测经ATRA处理后,K562细胞表面抗原CD71及CD13表达水平的变化;应用RNA/蛋白带移动分析方法 (RNA/protein band-shift assay)检测铁调节蛋白(IRP)的结合活性;采用放射免疫分析法测定K562细胞内铁蛋白含量的变化;采用RT-PCR方法对ATRA处理的K562细胞H-Fn、TfR及IRP2 mRNA水平的表达进行检测,同时对RT-PCR产物克隆测序。结果 K562细胞经ATRA诱导,出现粒系分化的形态学特征,中幼粒细胞及以后阶段的细胞数明显增多。细胞表面抗原CD71下降而CD13表达升高。ATRA处理组的H-Fn mRNA水平较对照组升高,而IRP2及TfR mRNA减少;处理组IRP结合活性较对照组明显下降,但铁蛋白含量升高。结论 ATRA诱导K562细胞向粒系方向分化过程中,伴有一系列铁代谢相关基因表达产物的改变,其上游调控机制有待进一步探讨。
Objective To investigate the effects of all-trans retinoic acid (ATRA) on expression of iron-related genes (KFH, TfR and IRP2) in K562 leukemia cells and their correlation. Methods Four kinds of staining methods of benzidine, Wright’s, non-specific esterase and nitro-tetrazolium blue were used to observe the differentiation characteristics of ATRA on K562 cells. Flow cytometry was used to detect the expression of ATRA on K562 cells The expression of antigens CD71 and CD13 were detected. The binding activity of iron regulatory protein (IRP) was detected by RNA / protein band-shift assay. The content of ferritin in K562 cells was determined by radioimmunoassay The mRNA expression of H-Fn, TfR and IRP2 in ATRA-treated K562 cells was detected by RT-PCR. The RT-PCR products were cloned and sequenced. Results The K562 cells were induced by ATRA, and the morphological features of myeloid differentiation were observed. The number of myeloid cells and their subsequent stages increased significantly. Decreased cell surface antigen CD71 and increased CD13 expression. The level of H-Fn mRNA in ATRA-treated group was higher than that in control group, but IRP2 and TfR mRNA were decreased. The binding activity of IRP in treatment group was significantly lower than that in control group, but the content of ferritin was increased. Conclusion ATRA-induced K562 cells to the direction of myeloid differentiation, accompanied by a series of iron metabolism-related gene expression changes, the upstream regulatory mechanism needs further study.