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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。
Objective: To investigate the effect of 5-Fu on the hematopoietic growth factor gene expression induced by Egr-1 promoter in the treatment of hematopoietic injury after chemotherapy. METHODS: Flt3 ligand (FL) and pCIneo eukaryotic expression vector (Egr-EF) carrying the Egr-1 regulatory sequence were constructed and transfected into bone marrow stromal cell line HFCL by lipofectamine and named as HFCL / EF; positive cells expressing EGFP green fluorescence were observed by flow cytometry and inverted fluorescence microscope; the effect of 5-Fu on FL expression in HFCL / EF cells was detected by RT-PCR, Western blot and ELISA; Flow cytometry 5-Fu-treated HFCL / EF cell supernatant on CD34 + cells and CFU-GM proliferation; the use of reactive oxygen species inhibitor N-acetylcysteine identified chemotherapy through reactive oxygen species-induced Egr-1 promoter CArG sequence regulation of downstream genes Specificity of expression. Results: The bicistronic gene expression vector (Egr-EF) with Egr-1 regulatory sequence was constructed. The content of FL in culture supernatants of HFCL / EF cells treated with 5-Fu was significantly higher than that without 5-Fu (P <0.01). In 5-Fu-treated HFCL / EF cells, EGFP activity, FLmR-NA and FL protein expression were enhanced, and N-acetyl Cysteine significantly reduced FL content. Conclusion: The hematopoietic growth factor gene expression regulated by Egr-1 promoter induced by 5-Fu has a certain recovery effect on the hematopoietic injury after chemotherapeutic drug treatment.