Gene Transfer Of Naked Plasmid Encoding For RhVEGF In Ischemic TRAM Flap

Backgroud and ObjectsTransverse recrus abdominis musculocutaneous flap(TRAM) is used very popular in plastic and reconstructive surgery of soft tissues , especially in breast reconstruction . Its relatively high rate of skin necrosis always bothered the doctors , so further research is required to overcome this problem . A lot of works have been done to increase blood supply resulting in decreased rate of skin necrosis . Therapeutic angiogenesis has been studied widely in the treatment of vascular insufficiency. VEGF is a potent and specific angiogenic factor . It has an important significance in the treatment of ischemic flaps . Gene therapy have emerged as promising modality for the revascularization of tissues . It has been proved that delivery of VEGF gene in animal model to treat myocardial ischemia and hindlimb ischemia can induce angiogenesis , improve hemeodynamics , increase blood supply . Researchers providing plasmid coding for rhVEGF to the vessel pedicle can increase the survival area of ischemic flap . Conclusively , there have been done little about gene therapy of ischemic flaps .Objects of the following investigation : (1) To design a stable ischemic TRAM flap model ; (2) To estimate the significance of rhVEGF gene to the survival of ischemic TRAM flap and to the microvessel density; (3) To investigate the relativity between VEGF protein expression and survival area of flap or subcutaneous MVD.Methods(1)Plasmid extraction , bacteria transferred with plasmid coding for aimed gene are amplified and extracted for plasmid by alkali.(2)The animals are devided to four groups.They are PcDNAVEGF , VEGF , PcDNA , saline.(S)Operation of TRAM flap , inferior TRAM flap with unilateral inferior rectus muscle pedicle .7days later , the photoes of the flaps are taken , then input into the computer . The amount of viable tissue within the flap are measured by AutoCAD .(4)VEGF analysis ,serum samples are measured for VEGF by VEGF ELLSA kit before , 1 day after , 3 days after , 5 days after , 7 days after and 9 days after administration of plasmid coding for rhVEGF(5)The expression of VEGF in skin was detected by immunohistochemical staining of streptavidin-peroxidase(S-P) method.(6)Microvessel density, microvessels in per mm2 subcutaneous tissues of the slice by staining with HE was counted .Results(l)The amount of viable tissue of TRAM flap model:3.61 ?1.06cm2.(2)The amount of viable tissue is significant different between group PcDNAVEGF compared with group saline, group PcDNAVEGF vs group PcDNA(P<0.01) , and group VEGF vs group saline(P<0.05).(3)The level of VEGF in the serum does not increase after administration of PcDNAVEGF .(4)The microvessel density is significant different between group PcDNAVEGF compared with group saline(P<0.05), group PcDNAVEGF vs group PcDNA(P<0.05), and group VEGF vs group saline(P<0.05).(5)In group PcDNAVEGF , the dark brown antigen-antibody complex deposited in the plasma of endothelial cells . Sometimes a band of dark brown antigen-antibody complex surrounding the vessel lumen can be seen. Similar deposition was not seen in skin without administration of PcDNAVEGF .Conclusion(1)The directly subcutaneous transducted rhVEGF gene could be expressed to biologic active VEGF.(2)The directly subcutaneous transducted rhVEGF gene could increase MVD.(3)The directly subcutaneous transducted rhVEGF gene could increase the amount of viable tissues within the TRAM flap .