Radioprotective effects of the expression of FLT3 ligand regulated by Egr-1 regulated element on radiation injury of SCID mice

Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal ...

Objective: In order to explore the radioprotective effects of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods:The human FL (Flt3 ligand) cDNA and EGFP (enhanced green fluorescent protein) cDNA were linked together with IRES and then inserted into the eukaryotic expression vector pCI-Egr, which was constructed by substituting CMV promoter in pCIneo with the Egr-1 promoter (Egr-EF). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin. The transduced cell clones (HFCL/EF) had been selected by the addition of G418. The cells were exposed to γ-radiation by ^(60)Co source for 0.5-20Gy. The expressions of transduced cells were detected with FACS, Northern blot ELISA and CFU assay. The HFCL/EF and CD34^+cells from human umbilical cord blood were one after the other transplanted i. v. into sublethally irradiated severe combined immunodeficient (SCID) mice. The white blood cell amount in peripheral blood and human cell engrafted in recipent mice were detected by flow cytometry and CFU-GM etc. Results: The activity of EGFP in transduced cells increased by 3.1 fold as compared to non-transduced cells at 18h after exposure to 2.5Gy. The amounts of secreted FL in serum-free supernatants of Egr-EF increased by 605. 46±107.21pg/ml, which were significantly higher than the control group (214.45±35.61pg/ml). The effects of FL in HFCL/EF cultural supernatants on expansion of CD34^+ cells derived from cord blood in the presence of SCF, IL-6 and IL-3 were also studied. The results showed that at day 10 of culture the number of CD34^+ cells increased by 173.09±11.58×10~3/ml, which was significantly higher than that of non-radiation group (68. 04±13.73×10~3/ml). It showed that radiation can enhance the ability of the supernatants containing FL of HFCL/EF to expand early hematopoietic progenitor cells and protect hematopoietic cells from radiation-injury effects. The HFCL/EF and CD34^+cells from human umbilical cord blood were one after the other transplanted i.v. into sublethally irradiated severe combined immunodeficient (SCID) mice. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, while no significant differences were found for CD45^+、CD34^+ cells in bone marrow cells. In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-drived expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the white blood cell at early stage after radiation, without significant differences being found for CD45^+、CD34^+、CFU-GM and marrow nucleared cells in bone marrow cells. Conclusions: The results suggested both in vivo and in vitro use of the gene therapy of FL gene regulated by Egr-1 promoter could protect hematopoiesis from irradiation-induced damage.