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目的 探讨红细胞分化相关因子 (EDRF) 1基因在人红白血病 (HEL)细胞中珠蛋白表达的影响。方法 构建EDRF1真核正义和反义表达载体 ,转染HEL细胞并筛选稳定表达细胞克隆。然后利用Northernblot和逆转录聚合酶链反应 (RT PCR)的方法观察红系标志基因表达水平的改变 ,应用凝胶阻滞电泳 (EMSA)的方法观察红系转录因子DNA结合活性的改变。结果 与对照相比 ,EDRF1过表达的HEL细胞中α 珠蛋白合成明显增加 ,EDRF1反义表达载体转染的HEL细胞中的α 、γ 珠蛋白合成下调 ,而红细胞生成素 (EPOR)表达水平没有明显改变。红细胞特异的转录因子GATA 1和NF E2mRNA的表达在转染前后没有明显改变。GATA 1转录因子的DNA结合活性在反义表达载体转染的HEL细胞中受到明显的抑制 ,而红系核因子 (NF E) 2的转录活性则没有明显的改变。结论EDRF1下调珠蛋白的合成是通过抑制红系特异的转录因子GATA 1的DNA结合活性实现的。
Objective To investigate the effect of erythroid differentiation-related factor (EDRF) 1 gene on globin expression in human erythroleukemia (HEL) cells. Methods EDRF1 eukaryotic sense and antisense expression vectors were constructed and transfected into HEL cells and screened for stable cell clones. Northern blot and reverse transcription polymerase chain reaction (RT PCR) methods were used to observe the changes of the expression level of erythroid marker genes. Gel-block electrophoresis (EMSA) was used to observe the changes of DNA binding activity of erythroid transcription factors. Results Compared with the control, α-globin synthesis was significantly increased in EDRF1-overexpressing HEL cells, α,γ-globin synthesis in HELRF-transfected HEL cells was down-regulated, and erythropoietin (EPOR) expression levels were not Obviously changed. The expression of red blood cell-specific transcription factors GATA 1 and NF E2 mRNA did not change significantly before and after transfection. The DNA binding activity of the GATA 1 transcription factor was significantly inhibited in the HEL cells transfected with the antisense expression vector, while the transcriptional activity of the erythroid nuclear factor (NF E) 2 was not significantly changed. Conclusion Down-regulation of globin by EDRF1 is achieved by inhibiting the DNA-binding activity of the erythroid-specific transcription factor GATA 1.