氟尿嘧啶诱导Egr-1启动子调控造血因子基因表达对造血恢复的影响  

Effect of Fiuorouracil-inducible GM-CSF gene therapy regulated by Egr-1 promoter on chemotherapeutic hematopoietic damage of tumor-bearing mice

作　　者：杜楠[1] 裴雪涛[2] 肖文华[1] 孙君重[1] 付艳[1] 赵晖[1] 王希良[1] 

机构地区：[1]解放军总医院第一附属医院肿瘤科,北京100037 [2]解放军总医院第一附属医院军事医学科学院9所,北京100037

出　　处：《中华医学杂志》2009年第18期1281-1286,共6页National Medical Journal of China

基　　金：国家自然科学基金（39900040）;解放军总医院第一附属医院新技术重大基金（ZD200502）,

摘　　要：目的探索5-氟尿嘧啶（5-Fu）诱导EgT-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GM—CSF和EGFP双顺反子基因pCIneo真核表达载体（Egr-EG）；通过脂质体转染骨髓基质细胞系HFCL，挑出C418抗性的阳性克隆（HFCL／EG）；采用流式细胞仪和倒置荧光显微镜观察EGFP绿色荧光表达的阳性细胞；在加入5-Fu的HFCL／EG细胞培养体系中，采用RT-PCR、Western印迹和ELISA检测GM—CSF的表达；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。将HFCL／EG细胞输入荷瘤的重症联合免疫缺陷（SCID）小鼠体内，对其实施5-Fu化疗，观察外周血象动态改变和对肿瘤的影响。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）；在HFCL／EG细胞中证实有外源性基因EGFP和GM-CSF的表达，在5-Fu处理后HFCL／EG细胞培养上清液GM—CSF含量较未加5-Fu组明显增高（P〈0．01）；RT-PCR和Western印迹显示化疗后HFCL／EG细胞GM-CSF mRNA和GM—CSF蛋白表达增强。在5-Fu处理的HFCL／EG细胞中，N-乙酰半胱氨酸明显减少GM—CSF含量。实验组与对照组相比外周血白细胞下降幅度明显减轻，恢复加快，而各组间肿瘤抑制率与化疗组相关，而与外源基因表达无明显的差异。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后的造血损伤具有一定的恢复作用。Objective In order to explore the regulating 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 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 transferred into human bone marrow stromal cell line HFCL by lipofectinTM. The transfected cell clones （HFCL/EG） have been selected by the addition of C,418. The cells are exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EG cells was detected by flow cytometry. The post-chemotherapeutical expression of GM-CSF in HFCL/EG was confirmed with ELISA and Western blot and RT-PCR respectively. The effect of N-acetylcysteine （ a free radical scavenger） on GM-CSF production post-exposure to 5-Fu was examined. The HFCL/EG cells were transplanted intravenously into B16 melanoma in C. B-17 combined immunodeficient （SCID） mice. 5-Fu was given i.p. at Day 3. The white blood cell number in peripheral blood, the expression of EGFP and GMCSF and in human stromal cell engrafted in recipient mice were detected by flow cytometry and RT-PCR respectively. Tumor volume in tumor-bearing mice was calculated. Results The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-Fu increased ascompared to non-5-Fu group with flow cytometry, RT-PCR and ELISA respectively. N-acetylcysteine significantly decreased the concentration of GM-CSF in HFCL/EG cells treated with 5-FU. In contrast to two control groups, HFCL/EG （Egr-1 regulatory element-derived expression of GM-CSF gene therapy） resulted in a proportionally obvious increase in the number of white blood cell after chemotherapy and no significant difference was found for tumor inhibition in recipient mice. Conclusions These in vitro data provide an exper

关 键 词：启动区(遗传学) 5-氟尿嘧啶 粒-巨噬细胞集落刺激因子 骨髓基质细胞 活性氧 

分 类 号：R686[医药卫生—骨科学]