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的保护作用。方法 本实验将携带Egr 1调控序列的FLT3配基 (FL)和EGFP基因双顺反子载体 (Egr EF)转染骨髓基质细胞系HFCL ,采用流式细胞仪、RT PCR、ELISA及细胞增殖法等观察细胞受照后Egr 1调控元件诱导的FL表达及促进造血细胞的增殖作用。 结果 在转染细胞HFCL/EF细胞中证实有外源性基因的整合和表达 ,在 2 5Gy辐射后 16h的细胞培养上清液中表明FL含量较照射前明显增高 ((P <0 0 1) ,) ;辐射后 10dHFCL/EF培养上清液对CD34+造血祖细胞的作用较辐射前具有明显的扩增作用 ((P <0 0 1) ,)。结论 在辐射后Egr 1启动子调控的FL基因表达明显增高并具有保护造血作用。
Objective\ To explore the regulatory effects of radiation-inducible gene on the expression of FLT3 ligand genes. Methods\ The human EL cDNA and EGFP cDNA were linked together with IRES and then inserted into the expression vector pCI-Egr-1, which was constructed by substituting CMV promoter in pCI-neo with the Egr-1 promoter(Egr-EF).The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin TM .The cells were exposed to γ-radiation from 60 Co source at 0 5-20Gy.The contents of green fluorescence in HFCL/EF cells were detected with FACS.The expression of FL in HFCL/EF cells was confirmed with RT-PCR and ELISA respectively.The effects of FL in HFCL/EF cultural supernatants on expansion of CD34 + cells were also studied. Results \ The amounts of secreted FL in serum-free supernatants of Egr-EF were significantly higher than in the control group.EGFP and FL cDNA were successfully integrated and expressed in the cells,which were confirmed by FACS,RT-PCR and ELISA analysis.On day 10 of culture the number of CD34 + cells was significantly higher than that of the non-radiation group. Conclusion \ These in vitro data provide an experimental basis for the in vivo use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from radiation injury.\;
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
2000年第5期306-309,共4页
Chinese Journal of Radiological Medicine and Protection
基金
国家自然科学基金!资助项目 (3990 0 0 40 )
国家杰出青年自然科学基金!资助项目 (3982 5 111)
