Experimental Reasearch Of The Influence Of VEGF Gene Therapy Following Electric Pulse On The Survival Of Random Flap In The Rats

Purpose:To study the gene expression and the angiogenesis effect of subcutannous injection of VEGF plasmid following electric pulse and their influence on the survival of random flap in the rat and evaluate safty of this gene therapy protocol.Methods:Construct a mammalian expression vector PCMV-MCS-VEGF-IRES2-EGFP. Mediate transient transfection of PCMV-MCS-VEGF-IRES2-EGFP to 293 cells with calcium phosphate in vitro.Transfer recombinant plasmid into rat flap by subcutannous injection following electric pulses.Observe the GFP mark gene expression in the skin through fluorescence microscope.compare the transfer efficacy of different dose VEGF plasmid.Observe the angiogenesis effects of VEGF gene through HE stain and α -actin stain.Design the model of superlong random flap in the rat and compare the effects of subdermal injection of plasmid with different dose following electric pulses on the survival of the flap.Observe the serum VEGF level in the gene therapy using subcutannous injection of plasmid following electric pulses by ELISA.Results:The eukaryon expression system PCMV-MCS-VEGF-IRES2-EGFP can express target protein in vitro.The recombinant plasmid was transferred into skin successfully through subcutannous injection following electric pulses.The expression of GFP can observed as early as six hours .It rise to its summit after 3 days of trsnsfer and last for 14 days.The higher dose plasmid have a relative higher transfer efficacy.Obvious angiogenesis effect can observed after 7 days of gene transfer.The gene therapy protocol using subcutannous injection of plasmid following electric pulses can increasing skin viability.But the different dose of plasmid have the same effect on the survival of a random flap.There is no change of serum VEGF level after subcutannous injection of plasmid following electric pulses.Conclusion:Subcutannous injection of plasmid following electric pulses can significantly increase the random flap viability.The higher dose plasmid have no significantly differents than lower dose plasmid on the survival of random flap.The gene...