摘要
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 study the effects of radiation inducible gene on the regulation of the expression of hematopoietic growth factor and the restoration of hematopoiesis.Methods Human GM-CSF cDNA and FLT3 Ligand(FL) were linked together with IRES and inserted into the expression vector pCI-Egr-1 which was constructed with the substitution of CMV promotor in pCIneo with Egr-1 promotor (Egr-FG). The vector was transferred into human bone marrow stromal cell line HFCL with lipofectinTM. The mRNA expression of the target gene in the cells were identified with RT-PCR. The amount of FL and GM-CSF in the supernatant of HFCL-EFG cell culture was determined with ELISA. CD34+cells isolated with MACS were cultured in the medium containing the supernatant of HFCL/EFG to observe its effect on the proliferation of CD34+cells.Results The gene expression vector (Egr-FG) initiated with Egr-1 regulation sequence was successfully constructed. It was confirmed that there were expression of exogenous FL and GM-CSF in HFCL-EFG cells. After 16 hours of 60Co irradiation, the amount of FL and GM-CSF in the serum-free supernatant of HFCL/EFG cell culture was increased respectively(P< 0.001).It was found that after irradiation, the supernatant of HFCL/EFG culture significantly increased its effect on the proliferation of CD34+cells.Conclusion These in vitro data provide an experimental basis for the in vivo use of gene therapy of hematopoietic growth foctor gene regulated by Egr-1 to protect hematopoiesis from ionizing radiation.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2000年第4期310-313,共4页
Journal of Third Military Medical University
基金
国家自然科学基金!资助项目(39900040)
国家杰出青年自然科学基金!资助项目(39825111)
关键词
辐诱导基因
造血生长因子
EGR-1启动子
s:radiation inducible gene
hematopoietic growth factor
gene therapy
radiation
bone marrow stromal cell
