Experimental Studies On The Enhancement Of TRAM Flap Survival In The Rat By Recombinant Adenovirus-mediated VEGF₁₆₅ Gene

Partial necrosis of pedicled skin flaps remains a significant problem in plastic and reconstruction surgery. Although the conventional TRAM flaps have a relatively high rate of skin necrosis which is about 6~28%, but it is regarded as an excellent flap for breast reconstruction. TRAM flap has become widely accepted in breast reconstruction. The further research is required to overcome this problem. Many method of treatment has being developed to increase the surviving area of skin paddle. It is includes that the technique of modified TRAM flaps, surgical delay, pharmacological delay, growth factors and gene therapy.Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific growth factor. It is an important inducer of angiogenesis. Previous studies evaluating the role of VEGF has indicated that locally applied exogenous VEGF protein can stimulate angiogenesis and improve skin and muscle flap survival. Although the results of these studies are promising, the effect attributable to direct application of VEGF in its soluble protein form could be transient.In recent years, gene therapy suing either naked plasmid DNA or viral vectors encoding for an angiogenic factor has become popular because it provides an effect of longer duration as opposed to the recombinant protein forms of angiogenic factor. In gene therapy, naked plasmid DNA or a viral vector containing the gene is delivered into a tissue and taken up by the host cells, leading to increased expression of protein factor. Use of virus-mediated gene therapy has advantage over the plasmidDNA, providing a higher rate of infection and efficiency.Several studies demonstrated the successful use of adenovirus vector encoding for VEGF in experimental and clinical settings. Lubiatowski et al. demonstrated that adenovirus-mediated VEGF 165 gene transfer into the subdermal space of the rat model significant induces the in epigastric flap survival. Patel et al. investigated the safety of direct myocardial administration of an adenovirus vector encoding VEGF121 in an attempt to revascularize the ischemic myocardium in a porcine model. The results of this study suggested that persistent expression of VEGF gene transferred with an adenovirus vector is not a significant safety risk.On the basis of these experimental results, in our study, we investigate that the effect of the subdermal space or/and rectus abdominis muscle adenovirus-mediated angiogenic gene therapy by using VEGF165 on the TRAM flap. In this study, we depend on the major strategy that transfer the VEGFi65 gene to target cells and enhance its expressing at the level of mRNA. It is the method that the replication deficient recombinant adenovirus-mediated VEGF165 gene was delivered into primary cultured myoblast, the subdermal space and rectus abdominis muscle of TRAM flap. To investigate the effect on the biological and angiogenesis of the myoblast and striped muscle transferred with Ad-VEGFi65 in vitro and in vivo. The effect and feasibility of the TRAM flap survival of local ischemic area in the rat by gene therapy with adenovirus-mediated vascular endothelial growth factor VEGF165 gene will be researched. The major work in our study is described as follows: 1. Amplification of the recombinant adenovirus-mediated VEGF165 gene and determination of viral particle titration.In our experimentation, the passage 3 of Ad-VEGFi65 and Ad-GFP, which already were identified, were amplified in QBI-293A cells. The viral particle titration of Ad-VEGFi65 and Ad-GFP were determined with the TCID50 method. Theviral particle titration of Ad-VEGFi65 is 5.0* 108 pfu/ml, and the viral particle titration of Ad-GFP is 6.4x108 pfu/ml after one amplification cycle. The shape of viral particle Ad-GFP and Ad-VEGFi65 were directly shown in electron microscopy after amplification with 40,000 times.2. Experimental studies of the recombinant adenovirus-mediated VEGF165 gene transfer to skeletal muscles myoblast in vitro.Studies show that recombinant adenovirus can transduce its DNA into nearly all cell lines, which lymphoid cell lines being among the most difficult to transduce. In our studies, we accomplished myoblast culture in vitro, in which originate from skeletal muscles cells of infant SD rat. The characteristic of myoblast were tested by Desmin, a-sarcomeric actin and skeletal myosin (alpha) immunohistochemical staining. In this condition, myoblast was selected as target cell that was transduced by recombinant Ad-VEGFi65 and Ad-GFP. The transduction infectivity of adenovirus to myoblast was tested by Ad-GFP, which is an adenovirus expressing green fluorescent protein. We found that the transduction infectivity of adenovirus to myoblast at 60MOI was from 67.73% and the efficiency increased with higher MOI reaching 97.33% at 120 MOI. The growth curve was used to determinate the cells growth rate and no significant difference was found between myoblast cultured group after infection with Ad-VEGFi65, Ad-GFP or 10% FBS-DMEM mock-infection. The VEGF165 protein expressing in myoblast were tested by VEGF immunohistochemical staining. Real-Time PCR were performed to determinate the level of exogenuos VEGF165 mRNA expressing. Myoblasts were infected with Ad-VEGFies at 60MOI and then the total cellular RNA was isolated at different times (2 to 5days) after infection. The maximal increase of exogenuos VEGF165 mRNA expression was observed at 5 day after infection. The result of experimental studies indicated that the exogenuos VEGF165 gene was successfully transduced intomyoblasts and then efficiently transcribed. Myoblast is an excellent expressing platform which was transduced with the exogenous gene.3. Experimental studies of the recombinant adenovirus-mediated VEGFks gene transfer to the TRAM flap in the rat in vivo.Thirty male Spraque-Dawley rats weighting 350 to 400g were used. Animals were housed in groups five by a choice at random. A total of 30 male SD rats were divided into five groups of 6 each, according to type of treatment. In groups I, II, and El, the TRAM flaps were pretreated with Ad-VEGFi65. GroupIV (the control gene encoding green fluorescent protein, Ad-GFP) served as adenovirus control. Group V (the control flaps) received DMEM. The rats were first anesthetized and previously marked 30mmx80mm TRAM flap outline with type 2 model (cephalically placed, inferiorly pedicled), which was based on the right inferior deep epigastric vessels (IDEV). In the TRAM flap with type 2 model, to delivery Ad-VEGFi65 into the subdermal space or/and rectus abdominis muscle of TRAM flap with 2.4x108pfu/ml titration. The final volume is 400ul or 800ul according to the different treatment groups. Ad-GFP is 800ul in volume as an adenovirus control. DMEM is 800ul in volume as a control flaps group. Operation of TRAM flap was performed in 7 days after injection with Ad-VEGFi6s, Ad-GFP and DMEM. The survival skin area from each experimental group was measured at 1 postoperative week. The tissue samples were taken from surviving regions. Frozen tissues sections were observed under laser cofocus fluorescence microscope. General sections were test with the following method: Staining with hematoxylin and eosin. CD34 immunohistochemical staining. VEGF immunohistochemical staining and VEGF mRNA in situ hybridization histochemical staining. Real-Time PCR were performed to determinate the level of exogenuos VEGF 165 mRNA expressing. The result of experimental studies indicated that the green fluorescence protein is positive expressing in frozentissues sections. It is proved that the Ad-GFP was successfully transduced into tissues and then efficiently transcribed. The survival skin area of Ad-VEGFi65 treatment groups is increased obviously. CD34 immunohistochemical staining is showed that the microvascular is increased. VEGF protein is positive expressing in striped muscle cells. The VEGF165 mRNA is shown positive expressing and orientating in striped muscle cells by method of VEGF-ISH with VEGF oligonucleotid probe. Real-Time PCR were performed to determinate the level of exogenuos VEGF165 mRNA expressing in tissues. All of the experimental result testified that Ad-VEGFi65 treatment groups were showed a statistically significant difference between the Ad-GFP and the DMEM treatment group (P<0.05). The result of experimental studies in vivo indicated that the VEGF 165 is increased the survival of TRAM flap obviously in the rat. Conclusion1. Ad-VEGFies and Ad-GFP were amplified in QBI-293A cells. We can get higher titration viral particle of Ad-VEGFi65 and Ad-GFP. In generally, the titration is higher than 1 x 108pfu/ml.2. The result of experimental studies in vitro indicated that the growth curve was used to determinate the cell growth rate and no significant difference was found that myoblast cultured group after infection with Ad-VEGFi65 and Ad-GFP at 60MOI. The exogenuos VEGF 165 gene was successfully transduced into myoblasts and then efficiently transcribed. Myoblast is an excellent expressing platform which was transduced with the exogenous gene in vitro.3. The result of experimental studies in vivo indicated that the Ad-VEGFi65 can be efficiently infected the tissue of TRAM flaps in the rats, significantly increased the expressing level of VEGF protein, stimulated angiogenesis in tissues, and improved survival area of the TRAM flap.