摘要
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 observe the differentiation inducing properties of the novel erythroid differntiation related gene EDRF1, and demonstrate its functioning pathway involved in regulation of α and γ globin gene expression. Methods EDRF1 sense and antisense constructs were transfected into HEL cells, then the expression of globin and erythropoietin receptor gene was identified by Northern blot analysis. RT PCR and electrophoresis mobility shift assay (EMSA) were carried out to monitor the expression and DNA binding activity of erythroid specific transcription factor GATA 1 and NF E2. Results In HEL cells with transfection of sense expression carrier, the experssion of EDRF1 mRNA was upregulated. In HEL cells with transfection of antisense expression carrier, the experssion of EDRF1 mRNA was downregulated. In HEL cells with overexpression of EDRF1, production of α globin was increased. In antisense EDRF1 overexpressed HEL cells, significant loss of α and γ globin mRNA synthesis was observed. The expression of erythropoietin receptor was unchanged in HEL cells transfected with EDRF1 sense and antisense expression carriers. No significant change was found in expression of transcription factors GATA 1 and NF E2 in HEL cells transfected with empty carrier, sense expression carrier, and antisense expression carrier. However, the transcription activity of GATA 1 was severely impaired. Expression of erythropoietin receptor gene was not influenced by EDRF1 gene overexpression. Conclusion EDRF1 regulates α and γ globin gene synthesis by modulating DNA binding activity of GATA 1 transcription factor. Overexpression and underexpression of EDRF1 do not significantly regulate the expression of GATA 1 and NF E2 mRNA. Monitoring the EPOR signal pathway is not the mechanism by which EDRF1 influences the differentiation of erythrocyte.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2001年第24期1512-1515,共4页
National Medical Journal of China
关键词
细胞分化
珠蛋白
转录因子
基因表达调控
白血病
Cell differentiation
Globin
Transcription factors
Gene expression regulation
