阿霉素诱导Egr-1启动子调控的造血因子GM-CSF基因表达对荷瘤小鼠造血功能恢复的影响

本研究探讨阿霉素（ADM）诱导早期生长因子（Egr-1）启动子调控的造血因子基因表达对荷瘤小鼠化疗后造血功能恢复的作用。将构建的携带有Egr-1启动子的粒-巨噬细胞集落刺激因子（GM-CSF）cDNA和增强型绿色荧光蛋白（EGFP）cDNA双顺反子真核表达载体导入基质细胞HFCL后，输入重症联合免疫缺陷（SCID）小鼠体内。实验小鼠随机分4组：①ADM诱导的HFCL／EG组（HFCL／EG＋ADM组），②ADM诱导的HFCL组（HFCL＋ADM组），③单纯输注HFCL／EG组（HFCL／EG组）和④单纯输注HFCL组（HFCL组），每组6只动物。观察外周血象动态改变，用流式细胞术检测eGFP^＋人基质细胞，用RT-PCR和Westernblot分别检测GM-CSFmRNA及其蛋白的表达。结果表明：化疗组与未化疗组相比外周血白细胞降低，而且下降幅度较低，恢复加快；各组间CFU-GM数无显著性差异；肿瘤抑制率与化疗相关，而与外源基因表达不相关；ADM处理后实验组小鼠骨髓可见绿色荧光阳性的基质细胞；RT，PCR和Westernblot显示GM-CSFmRNA和GM-CSF蛋白表达增强。结论：ADM诱导的Egr-1启动子造血因子基因疗法具有化疗后促进造血恢复作用。

Effect of Doxorubicin Induced GM-CSF Gene Expression Regulated by Egr-1 Promoter on Recovery of Hematopoietic Function in Bearing-melanoma Mice

This study was purposed to investigate the effect of hematopoietic growth factor expression regulated by Egr-1 promoter on the recovery of hematopoietic function in bearing-melanoma mice after chemotherapy with doxorubicin （ADM）. The human GM-CSF cDNA and enhanced green fluorescence protein （GFP） cDNA were linked together with internal ribozyme entry site （IRES） and then were inserted into the expression vector pCI-neo under control of the Egr-1 promoter （Egr-EG）. This vector was transduced into human bone marrow stromal cell lines HFCL by lipofectamine and was transfused in severe combined immunodeficiency （SCID） mice. The experimental mice were randomly divided into 4 groups （6 mice in each group） ： （ 1 ） HFCL/EG ＋ ADM group in which the HFCL/EG cells were transplanted intravenously in SCID mice bearing melanoma, ADM was given intraperitoneally after 3 days ; （ 2 ） HFCL ＋ ADM group in which the HFCL cells were transplanted intravenously, ADM was given intraperitoneally after 3 days; （ 3 ） HPCL/EG group in which HFCL/EG cells were transplanted alone; （4） HFCL group in which HFCL cells were transplanted alone. The dynamic change of peripheral blood picture was assayed by hemocytometer; the eGFP＊ human stromal cells were detected by flow cytometry; the expression of GM-CSF mRNA and protein were determined by RT-PCR and Western blot respectively. The results indicated that as compared with HFCL/EG and HFCL groups, the leukocyte count in HFCL/EG ＋ ADM group decreased, but decrease level was weaker than that in HFCL ＋ ADM group, meanwhile the recovery of leukocyte count was earlier than that in HFCL ＋ ADM group. The CFU-GM amount between 4 groups showed no significant difference. The detection results showed that the inhibitory rate of tumor was related to chemotherapy, but not to expression of exogenous gene; the eGFP^＋ stromal cells existed in bone marrow of mice treated with ADM. The RT-PCR and Western blot assays revealed enhancement of GM-CSF mRNA and protein. It is concluded that the ADM-inducible GM-CSF gene therapy regulated by Egr-1 promoter may promote the hematopoietic recovery after chemotherapy.