氟脲嘧啶促进Egr-1启动子上调人骨髓基质细胞GM-CSF的表达

5-fluorouracil enhances Egr-1 promoter upregulate expression of granulocyte-macrophage colony-stimulating factor in human bone marrow stromal cells

目的:探索氟脲嘧啶(5-FU)对Egr-1启动子上调人骨髓基质细胞造血因子GM-CSF表达的促进作用,以寻找促进化疗所致造血损伤恢复的方法。方法:构建携带Egr-1调控序列启动的GM-CSF和EGFP双顺反子基因的重组真核表达载体(pCIneo-Egr-1-EGFP-IRES-GM-CSF,Egr-EG),通过脂质体转染骨髓基质细胞系HFCL,挑出G418抗性的阳性克隆(HFCL/EG)。采用RT-PCR检测5-FU处理的HFCL/EG细胞GM-CSFmRNA表达,用FACS和倒置荧光显微镜观察5-FU诱导HFCL/EG细胞EGFP表达的阳性细胞。在加入5-FU的HFCL/EG细胞培养体系中,用ELISA方法检测GM-CSF的含量;将从脐血中分离的单个核细胞接种于5-FU处理后的HFCL/EG培养上清液培养基中,观察其对GM-CFU的增殖作用;采用活性氧抑制剂N-乙酰半胱氨酸检测5-FU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-EG,获得其转染细胞HFCL/EG。在5-FU处理的HFCL/EG细胞中,RT-PCR显示其GM-CSFmRNA表达增强,流式细胞术证实有EGFP的显著表达。在5-FU处理后,HFCL/EG细胞培养上清液GM-CSF含量和GM-CFU形成数量分别较未处理细胞能明显增高(P<0.01);在5-FU处理的HFCL/EG细胞中,N-乙酰半胱氨酸能明显减少GM-CSF含量(P<0.01)。结论:5-FU能促进Egr-1启动子上调人骨髓基质细胞GM-CSF基因的表达,从而对化疗后的造血损伤产生一定的恢复作用。

[ Abstract] Objective： To explore 5-fluorouracil-induced regulating effect of Egr-1 promoter on expression of granulo- cyte-macrophage colony-stimulating factor （GM-CSF） in human bone marrow stromal cells. Methods： The human GM- CSF cDNA and enhanced green fluorescent protein （EGFP） cDNA were linked together with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter（Egr-EG）. The vector was then transferred into human bone marrow stromal cell line HFCL by lipofection. The transfected cell clones （HFCL/EG） were selected by the addition of G418. The cells were exposed to the anticancer agent 5-fluorouracil （5-FU）. The activity of EGFP in HFCL/EG cells were detected by FACS. The amounts of GM-CSF in HFCL/EG postchemotherapy were determined with ELISA. The effects of GM-CSF in HFCL/EG cultural supernatants on expansion of CFU-GM derived from cord blood were also studied. The effect of N-acetylcysteine （a free radical scavenger） on GM-CSF production was examined following exposure to 5-FU. Results： We successfully constructed vector Egr-EG with Egr-1 promoter, and its transfeetant HFCL/EG was ob- tained. The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCIJEG cells exposed to 5-FU were increased compared to non-5-FU group. The content of GM-CSF in HFCL/EG cultural supernatants was signifi- can@ higher than that in the non-5-FU group （P 〈 0. 01 ）. N-acetylcysteine significantly decreased the content of GM-CSF produced by HFCL/EG treated with 5-FU （P 〈 0. O1 ）. Conclusion： 5-FU can enhance Egr-1 upregulate GM-CSF expression in human bone marrow stromal cells, and thus contribute to the recovery of hematopoietic function after chemotherapy.