The Effect Of VEGF And HO-1 Gene On Survival Of Distant Oversized Random Flap In Rats

Random flap is commenly used to wound covering, function reconstruction and appearance improvement. But the distant end of the flap which is beyond a certain length/width ratio can easily get necrosis.There are two critical success factors for flap grafting.One is the blood supply of the root flap.The other is neovascularization.If neovascularization is interrupted, survival area of the flap will be less.The surviving process of the distant end is similar with skin flap.As we all know, Vascular endothelial growth factor (VEGF), a mitogen that promotes vascular endothelial cells'proliferation and angiogenesis[16], is a 45-kDa glycoprotein secreted in the vascular wall by endothelial and smooth muscle cells. VEGF promotes angiogenesis in normal physiologic conditions such as wound repair and in pathologic conditions including atherosclerosis, diabetic retinopathy, tumor growth and metastasis formation.But in fact,vegf had disadvantages.Remote organizations of the flap suffered serious hypoxia and ischemic for a long time resulting in the injury of the vescular endothelial cells. There was not enough time for the endogenous secretion of VEGF. In order to compensate for disadvantage of VEGF, we adopted HO-1 which has the function of anti-oxidation, anti-inflammatory, anti-apoptotic for joint application.It can obviously enhance the ability of anti-hypoxia gaining time for secretion of vegf of the body, hoping increase the survival area of the remote ultra-long random flap.Several lines of evidence suggest that heme oxygenase-1 (HO-1) is a component of cellular defence mechanisms against oxidative stress-mediated injury.Heme oxygenase (HO) is the rate-limiting enzyme for heme degradation in mammalian cells. HO catabolizes cellular heme,which is a prooxidant, to carbon monoxide (CO), bilirubin and iron. Three HO isoforms have been characterized; each encoded by a different gene. An inducible form HO-1, is induced by a variety of stresses such as oxidized lipoproteins,cytokines, hemodynamic changes, angiotensin II and nitric oxide (NO) in vascular wall. HO-1 induction seems to function as an adaptive response against these injurious stimuli. Soares and colleagues have reported that has found that the expression of vegf and ho-1 can stimulate the formation of a positive feedback loop,that is the expression of vegf can upregulate the secreation of ho-1, and vice versa.Gene expresson includes virus expression vector which consists of retroviral RNA, adenovirus DNA and adeno-associated virus and non-virus expression which involves plasmid, liposomes, naked DNA and gene gun.Currently, adenovirus vector is the most convenient and most widely used.There are plenty of advantages of adenovirus.①It has wide host range and low pathogenicity for people.②It can infect the proliferation and non-proliferating cells and have expression of genes.③Have high proliferation and titer.④Not integrated into the chromosome and no insert Mutagenicity.⑤Expression of multiple genes at the same time. Precisely because of the advantage above, adenovirus is widely used in gene transduction in vitro, vaccination in vivo and gene therapy.This study was conducted to investigate the united biological activity of VEGF and HO-1 expression. Target cells were transfected by adenovirus which can sustainly express the target protein. It can overcome the disadvantages of traditional biological agents such as short half-life, low activity and high cost. There were no relevant reports at home and abroad about increasing survival area of remote random flap through adenovirus VEGF and adenovirus HO-1. We designed this experiment subjects and carried out three-part of this experimental study.1. Construction of Ad5 repilication-deficient adenovirusAd-VEGF165 and Ad-HO-1To construct the new recombinant adenoviruses Ad-VEGF165 and Ad-HO-1 by homologous recombination, the plasmid PDC315-VEGF and PSUCMV-HO-1 were co-transfected with pBHGE3 into 293 cells through Lipofectamine2000 respectively. The recombinant adenoviruses were confirmed by PCR analysis using specific primers, amplified at a large scale and purified by cesium chloride gradient centrifugation. The viral titer of Ad-VEGF165 achieved1×1010 pfu/ml while Ad-HO-1 7.5×109 pfu/ml with TCID50 method.2. Biological activity of Ad-VEGF165 and Ad-HO-1 in vitroFor the purpose of comparing the infection capability of adenoviruse, the normal cells BJ were cultured and transfected with Ad-EGFP. The results were observed under the fluorescence micro- scope. The transfection and protein expression of VEGF gene was determined by ELISA. Because the expression of HO-1 gene in recombinant adenoviruse Ad-HO-1 infected host cells was demonstrated to locate to the mitochondrion .So the protein expression of HO-1 gene was determined by western blot and imunohistochemisty. The results of ELISA, western blot and imunohistochemisty all show that Ad-VEGF165 and Ad-HO-1 expresse large numbers of taget proteins in BJ cells. MTT assay was performed to determine the safty of Ad-VEGF165 and Ad-HO-1 at various viral MOIs. The transfection was demonstrated by flow cytometry. The results showed that the growth and proliferation of BJ cells transfected by Ad-VEGF165 and Ad-HO-1 were the same as the normal BJ cells.So Ad-VEGF165 and Ad-HO-1 were safe to use.3. Antitumor efficacy of SG235-TRAIL in vivo25 female Sprague-Dawley rats weighing between 250 and 350g were used for this experiment. One randon flap was made on each dorm of the Sprague-Dawley mice(2 flaps each mouse).Then mice were allotted randomly into five groups equally: Ad-VEGF165+Ad-HO-1(A group),Ad-VEGF165(B group),Ad-HO-1(C group),Ad-11B-GFP(D group),Ad-buffer(E group) (n=5 mice/10 flap/group). Each flap underwent 10 dots injections in the deep fascia, with total dosage of 109 pfu per flap. After 10 days,all animals were killed and 3 small tissues of flap were removed and fixed in 10% neutral formaldehyde for 6h and paraffin-embedded, and 5-μm-thick consecutive sections were cut for H&E staining, immunohistochemistry and MVD counting. VEGF165 and HO-1 protein were detected by immunohistochemistry.The expression of CD31 was much more in A group than other 4 groups.Compared with the control group and other treatment groups, survival rate of combination use of Ad-VEGF165 and Ad-HO-1 was much higher.Obvious necrosis was observed by routine pathologic examination in the end of the long flap in the certain groups.Collectively, we have constructed 5-type repilication-deficient adenovirus Ad-VEGF165 and Ad-HO-1,whose joint application has obviously increased the survival area of remote random flap.The project has given full play to HO-1 for its ablity of anti-oxidation, anti-inflammatory, anti-apoptotic and synergy with VEGF(165). It provides a new clinical treatment strategy and new ideas to improve the aspect ratio of flap and its survival rate.