The Investigation Of HGM-CSF Gene Transfer By In Vivo Electroporation To Remedy The Radiation Injury Of Mice

Object: Granulocyte-Macrophage Colony Stimulating Factor(GM-CSF) have the function of stimulating haemopoietic stem cell and haemopoietic ancestral cell differentiate and generate to medullary system cell. It has been used as one kind of fairly effective cytokine medicine for acute radiation injury. But as the defect of shorting biologic half-life, serious side reaction and the hard dosis controling, the therapeutic action of GM-CSF have been limited. To explore a new gene delivery method for treating radiation damage, we delivery the hGM-CSF gene into mice by in vivo electroporation.Materials and Methods: Clone the objective gene from prokaryotic vector by PCR, insert this gene to eukaryotic expression vector pEGFP-N1 by changing the two sides restricted enzyme site of the objective gene, in this way, we construct the recombinant eukaryotic expression plasmid pEGFP-N1-hGM-CSF. Using the in vivo gene delivery device to transfer the hGM-CSF gene into the mice muscle fibrocyte , make the expression of hGM-CSF protein to be persistent. Irradiate the gene transfer group and irradiate group withγray and neutron, to explore the effect of hGM-CSF gene transfer in vivo for treating the acute radiation injury by observing the death rate, the hematocyte population, the proliferation rate and apoptosis rate of bone marrow cell and spleen and thymus lymphocyte, the pathological change of liver,kidney and orchic.Results: The cDNA sequence of pEGFP-N1-hGM-CSF is coincident with Gene Bank certifying by gene sequencing. The transduction efficiency of in vivo electroporation increase obviously. In acute radiation injury ofγray, the death quantity of hGM-CSF gene transfer group is lower obviously than radiate group, as the means of total white cell count. The count of white cell can recovery to unirradiate level in the 28th day. The proliferation rate of bone marrow cell is also higher than radiate group obviously (p<0.05), and lasting for higher level(about 80%-95% of unirradiate group) all the time. The liver and kidney tissue of gene transfer group have a slight hydropsia, necrosis and hyperaemia to control group. And the duration of recovery is also shorter than control group. In acute radiation injury of neutron, the death rate of hGM-CSF gene transfer group is lower obviously than radiate group, the decrease degree of white cell count is lessen either(p<0.05),the apoptosis rate of bone marrow cell and spleen and thymus lymphocyte is also lower than radiate group.Conclusion:1. Construct the recombinant eukaryotic expression plasmid pEGFP-N1 -hGM-CSF, the plasmid can be used as the vector of iv vivo electroporation, and it can access to a high transduction efficiency.2. hGM-CSF gene electroporation iv vivo could decrease the mice death rate, alleviate the degree of white cell decreasing, elevate the proliferation activity of bone marrow cell, shorten the recovery duration of the liver and kidney tissue , relieve the degree of patho-damage in acute radiation injury ofγray.3. In acute radiation injury of neutron, hGM-CSF gene electroporation iv vivo could decrease the mice death rate obviously, decrease the apoptosis rate of bone marrow cell and spleen and thymus lymphocyte obviously also, alleviate the degree of white cell decreasing either.4. hGM-CSF gene electroporation iv vivo could be a new methond for treating acute severe radiation injury.