节点文献
氟脲嘧啶诱导Egr-1启动子转录调控Flt3配基的基因表达
Effects of fluorouracil-induced Flt3 Ligand gene expression transcriptional regulated by Egr-1 promoter sequences
【摘要】 目的:探讨5-氟脲嘧啶(5-Fu)诱导Egr-1启动子调控造血生长因子基因表达对化疗后造血损伤的恢复作用。方法:构建携带Egr-1调控序列启动的Flt3配基(FL)和EGFP双顺反子基因pCIneo真核表达载体(Egr-EF);通过脂质体转染骨髓基质细胞系HFCL并称为HFCL/EF;采用流式细胞术和倒置荧光显微镜观察EGFP绿色荧光表达的阳性细胞;采用RT-PCR、Western blot和ELISA检测5-Fu对HFCL/EF细胞FL表达的影响;流式细胞术检测经5-Fu处理的HFCL/EF细胞上清对CD34+细胞和CFU-GM的增殖作用;采用活性氧抑制剂N-乙酰半胱氨酸鉴定化疗通过活性氧诱导Egr-1启动子CArG序列调控下游基因表达的特异性。结果:构建了Egr-1调控序列启动的双顺反子基因表达载体(Egr-EF);经5-Fu处理后HFCL/EF细胞培养上清液较未加5-Fu组的FL含量明显增高并且对CD34+细胞和CFU-GM具有较强的扩增作用(P<0.01);在5-Fu处理的HFCL/EF细胞中,EGFP活性、FLmR-NA和FL蛋白表达增强,而且,N-乙酰半胱氨酸明显减少FL含量。结论:5-Fu诱导Egr-1启动子调控的造血生长因子基因表达对化疗药物处理后的造血损伤具有一定的恢复作用。
【Abstract】 AIM:To explore the regulatory effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil(5-Fu) on the expression of hematopoietic growth factor genes.METHODS:The human Flt3 Ligand(FL) cDNA and the enhanced green fluorescent protein(EGFP) cDNA were linked with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter(Egr-EF).The vector was transferred into human bone marrow stromal cell line HFCL.The transfected cells(HFCL/EF) were exposed to the clinically important anticancer agent 5-fluorouracil.The activity of EGFP in HFCL/EF cells was detected by FACS.The expression of FL in HFCL/EF postchemotherapy was confirmed by ELISA,Western blot and RT-PCR,repectively.The effect of FL in HFCL/EF cultural supernatants on the expansion of CD34+ cells and CFU-GM was studied.The effect of N-acetylcysteine(a free radical scavenger) on FL production following the exposure to 5-Fu was examined.RESULTS:The activity of EGFP and the amount of secreted FL in HFCL/EF cells exposed to 5-Fu increased compared to those in non-5-Fu group.The effects of FL in HFCL/EF cultural supernatants on the expansion of CD34+ cells and CFU-GM were more significant than those of non-5-Fu group.N-acetylcysteine significantly decreased the concentration of FL produced by HFCL/EF treated with 5-FU.CONCLUSION:The therapy of hematopoietic growth factor gene regulated by Egr-1 promoter can protect hematopoiesis from 5-Fu injury.
【Key words】 early growth response-1; 5-fluorouracil; Flt3 Ligand; bone marrow stromal cell; radical oxygen intermediate;
- 【文献出处】 细胞与分子免疫学杂志 ,Chinese Journal of Cellular and Molecular Immunology , 编辑部邮箱 ,2009年05期
- 【分类号】R96
- 【下载频次】98