The Role Of PcDNA3/HVEGF165Naked Plasmid In Promoting The Healing Of Full-thickness Skin Wounds Of Diabetic Mice

Part1The Establishment Of The Diabetic Mice Full-thickness Skin Defect ModelObjective: In this study,replication of full-thickness skin defect model on the basis of ICR mice which induced by streptozotocin(STZ).We observed changes in the wound healing time and the level of expression of VEGF,to explore a new method of building diabetic mice wound healing model for the follow-up study to provide a reliable animal model.Methods:36four weeks old ICR mice were randomly divided into two groups. including normal control(ND group)12, high fat diet+STZ group(HS group)24.Two groups of mice were taken weekly measurement of body weight in4to15weeks of age and weekly fasting blood glucose levels on4,8,9and10to15weeks of age.4weeks after the feeding, the HS mice by intraperitoneal injection of STZ80mg/Kg induction of diabetes, fasting trace glucose value≥II.lmmol/L mice were included in the diabetic group(DM group).A copy of the full-thickness skin defect model after the diabetes modeling successfully after4weeks(13weeks).After surgery were wound dressing,photography,drawing.The NIH Image analysia software for the determination of the wound healing rate,Real time-PCR detection wound organization mVEGF-A expression.Results:After STZ induced by intraperitoneal injection, the DM group, blood glucose was significantly increased up to18.76+1.75mmol/L, compared with the ND group7.30±0.62mmol/L.There were significant differences (P<0.05), at the same time observed modeling the increase in mouse urine,weight loss, diabetes was100%into a mold,and high blood sugar levels at least for6weeks. The wound healing of DM group delayed compared with the ND group,7,10,14days after surgery were statistically significant (P<0.05),while7th postoperative day.the expression levels of mVEGF-A mRNA of DM group were also lower than the ND group(P<0.05).Conclusions: High fat diet+STZ induced mice diabetes model approach practicable, modeling a success rate of100%,and high blood sugar steady state can be maintained for at least6weeks. The diabetic mice full-thickness skin defect model wound healing rate than normal mice significantly reduced, expression of mVEGF-A is also reduced.in line with the characteristics of wound healing can be applied to animal studies.PartⅡThe Role Of pcDNA3/hVEGF165Naked Plasmid In Promoting The Healing Of Full-thickness Skin Wounds Of Diabetic MiceObjective:The full-thickness skin defect model of diabetic mice constructed by the previous experimental has the characteristics of the diabetic wound healing delay.Therefore, in this part of the experiment.we will be using the model to observe the role of recombinant plasmid pcDNA3/hVEGF165promote wound healing.Methods:36four weeks old ICR mice were randomly divided into2groups were given normal rat food (group C,n=12) or15%lard-enhanced high fat rat grain feeding(DM group,n=24). After high-fat rat food fed for4weeks(8weeks old).the high-fat rat food kept in24mice by intraperitoneal injection of STZ80mg/Kg induced diabetes model. Fasting plasma glucose≥ll.lmmol/L as the inclusion criteria of the diabetic mice.Then were randomly divided into pcDNA3empty plasmid+full-thickness skin defects(Group P), pcDNA3/hVEGF165+full-thickness skin defect group(Group V).Diabetes modeling success after4weeks(13weeks), two groups of mice.a copy of the full-thickness skin defect model.C, P, V groups had a margin of intradermal injection of200ul PBS,60ug/100ul pcDNA3empty plasmid,60ug/100ul pcDNA3/hVEGF165plasmid on the back of each mice immediately after full-thickness skin wounds modeling. Postoperative day,3,7,10,14days were wound fixed focus camera,3,7,10,14days wound dressing.7and14days drawing.The NIH ImageJ image analysis software for the determination of wound healing rate.Real-time PCR detect the expression of hVEGF-A, mVEGF-A, mCOL-Ia, mVCAM of wound tissue, and histopathologic examination.Results:Compared with group C, the levels of blood glucose of group P and V are significantly high (P＜.01) but the blood glucose between group P and V have no significant difference, and the hyperglycemic state maintain at least the end of the experiment (ie,14days after surgey). Compared with group C, the wound healing rate of group P decreased at day7(P<0.05) and the wound healing rate at day10,14was significantly lower((P<0.01, Table3).Group C and group V.the wound healing rate was no significant differences at each time point (P>0.05).Compare with group P, the wound healing of group V was more faster, and have a statistically difference after10days of surgery. The relative expression of hVEGF-A of group V was significantly higher than group C and P. and has a statistically significant difference after7,14days of surgery (P<0.05).The expression of mCOL-Ia and mVCAM mRNA of group V and P were lower than group C in wound tissue of each mice,but no significant difference (P>0.05). The expression of mCOL-Ia and mVCAM mRNA of group V was higher than group P,but no significant difference (P>0.05).Conclusions:The injection of pcDNA3/hVEGF165naked plasmid allows hVEGF165successfully transfected, and contribute to diabetic mice wound healing. Its mechanism wound improve the expression of hVEGF165directly in the wound tissue of mice. And we know that the expression of mVEGF-A also improvement follow the transfer of hVEGF165.