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目的探索辐射诱导基因调控元件启动的造血生长因子表达及其对造血恢复的作用。方法该实验将携带Egr 1调控序列启动的FLT3配基(FL)和GM CSF双顺反子表达载体(Egr FG)导入骨髓基质细胞系HFCL(称为HFCL/EFG)。采用RT PCR、ELISA及细胞增殖法观察FL和GM CSFcDNA在转染细胞中的表达及保护造血作用。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 FG) ;在HFCL/E FG细胞中证实有外源性FL和GM CSF基因的整合和表达 ,在辐照后16h的HFCL/EFG细胞培养上清液中 ,FL和GM CSF含量较照射前明显增高(P<0.01) ;同时证实辐射后HFCL/EFG培养上清液对造血祖细胞有明显扩增作用 ;共培养的骨髓有核细胞和HFCL/EFG经照射后7d计数非粘附细胞 ,HFCL/EFG组较对照组明显增加(P<0.05)。结论辐射诱导基因Egr 1调控序列启动的造血生长因子基因表达载体在辐射后表达明显增高 ,在体外对辐射后的造血细胞具有保护作用。
Objective To explore the expression of hematopoietic growth factor activated by radiation-induced gene regulatory elements and its effect on hematopoietic recovery. Methods FLT3 ligand (FL) and GM CSF bicistronic expression vector (Egr FG), which were driven by Egr 1 regulatory sequence, were introduced into the bone marrow stromal cell line HFCL (called HFCL / EFG) in this experiment. The expression of FL and GM CSFcDNA in transfected cells and the protection of hematopoiesis were observed by RT PCR, ELISA and cell proliferation assay. Results The Egr 1 gene promoter promoter (Egr FG) was constructed. The integration and expression of exogenous FL and GM CSF genes were confirmed in HFCL / E FG cells. After 16 h of irradiation, HFCL / EFG cell culture supernatant, FL and GM CSF levels were significantly higher than before irradiation (P <0.01); the same time, HFCL / EFG supernatant was confirmed after irradiation on the proliferation of hematopoietic progenitor cells; co-cultured bone marrow Non-adherent cells were counted at 7 days after irradiation in nucleated cells and HFCL / EFG group, and increased significantly in HFCL / EFG group compared with control group (P <0.05). Conclusions The hematopoietic growth factor gene expression vector initiated by the radiation-inducible gene Egr 1 is significantly increased after radiation and has a protective effect on irradiated hematopoietic cells in vitro.