摘 要 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CSF mRNA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-CSF mRNA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 granulocyte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 transfectant HFCL/EG was obtained. The results indicated that the activity of EGFP and the amounts of secreted GMCSF in HFCL/EG cells exposed to 5-FU were increased compared to non5-FU group. The content of GM-CSF in HFCL/EG cultural supernatants was significantly 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<0.01). 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.

国家自然科学基金资助项目(No.39900040) ；解放军总医院第一附属医院新技术重大项目（ZD200502）