CAJ | 학술논문

目的观察EDRF1基因在HEL细胞中对红细胞终末分化和珠蛋白表达的扮演的角色.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Northern blot和反转录PCR (reverse transcription-polymerase chain reaction, RT-PCR)的方法观察红系标志基因的表达水平改变.凝胶阻滞电泳(electrophoresis mobility shift assay, EMSA)的方法观察红系转录因子的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,而红细胞生成素受体(erythropoietin receptor, ER)表达水平没有明显改变,这提示EDRF1并非通过调控ER信号通路而影响HEL细胞的红细胞分化.红细胞特异的转录因子GATA-1和NF-E2 mRNA的表达在转染前后没有明显改变.另外,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NF-E2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.
목적관찰EDRF1기인재HEL세포중대홍세포종말분화화주단백표체적분연적각색.방법구건EDRF1진핵정의화반의표체재체,전염HEL세포병사선은정표체세포극륭.연후이용Northern blot화반전록PCR (reverse transcription-polymerase chain reaction, RT-PCR)적방법관찰홍계표지기인적표체수평개변.응효조체전영(electrophoresis mobility shift assay, EMSA)적방법관찰홍계전록인자적DNA결합활성적개변.결과여대조상비,EDRF1과표체적HEL세포중α-주단백합성명현증가,EDRF1반의표체재체전염적HEL세포중적α、γ-주단백합성하조,이홍세포생성소수체(erythropoietin receptor, ER)표체수평몰유명현개변,저제시EDRF1병비통과조공ER신호통로이영향HEL세포적홍세포분화.홍세포특이적전록인자GATA-1화NF-E2 mRNA적표체재전염전후몰유명현개변.령외,GATA-1전록인자적DNA결합활성재반의표체재체전염적HEL세포중수도명현적억제,이NF-E2적전록활성칙몰유명현적개변.결론 EDRF1하조주단백적합성시통과억제홍계특이적전록인자GATA-1적DNA결합활성실현적.
Objective To further characterize the differentiation inducing properties of EDRF1 and demonstrate its functional pathway involved in regulation of globin gene expression. Methods By transfecting EDRF1 sense and antisense constructs into HEL cells, we identified the expression of globin and erythropoietin receptor genes by Northern blot analysis. RT-PCR and EMSA (electrophoresis mobility shift assay) were performed to monitor the expression and DNA-binding activity of erythroid specific transcription factors GATA-1 and NF-E2. Results It was shown that when EDRF1 was overexpressed, production of α-globin increased. In antisense EDRF1, overexpression of HEL cells, significant loss of α-, γ-globin mRNA synthesis was observed. The transcription of endogenous GATA-1 and NF-E2 mRNA expression were maintained at the same levels compared with control experiments. However, the transcription activity of GATA-1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. Conclusion The results suggested that EDRF1 regulated α- and γ-globin gene synthesis by modulating DNA-binding activity of GATA-1 transcription factor.