Studies on the expression of hematopoietic growth factors regulated by Egr-1 promoter

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. The cells were exposed to γ-radiation from 60Co 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