The Preliminary Study On The Treatment Of Skin Wounds In Type 2 Diabetes Mellitus Rat By Tissue Engineering Skin Transplantation

Objective:1. To establish the rat type 2 diabetes mellitus models and the skin wound model with type 2 diabetes mellitus.2. vascular endothelial growth factor (VEGF) gene was transfected into bone marrow mesenchymal stem cells (BMSCs), and combined the cells with Collagen sponge to construct tissue-engineered skin.3. The tissue-engineered skin was transplanted to heal the skin defect with type 2 diabetes mellitus, then the effect of the tissue-engineered skin was observed.Methods:1. The rabbit Type 2 diabetes mellitus models was induced by 2 month feeding with high glucose and grease and streptozocin injection. The blood fat and the insulin was detected at the time of 2 months feeding, and randomly selected 20% of the experimental animals to euglycemic clamp experiments。Then the skin defect was estabolished based on the type 2 diabetes model.2. BMSCs were obtained from SD rat and in vitro cultured. Then the subcultured BMSCs were transfected with VEGF, followed by incubation on collagen sponge construct the tissue-engineered skin.3. To build the diabetic rats medels with cutaneous chronic wounds.Four groups are determined according to different therapy:DM-blank groups (groups with non-theropy); DM-collagen group (groups with therapy of collagen); DM-MSCs groups (groups with therapy of tissue-engineered skin that constructed with collagen sponge and MSCs); DM-MSCs-VEGF groups (groups with therapy of tissue-engineered skin that constructed with collagen sponge and MSCs. These MSCs were transfected with VEGF). After the establishment of diabetes rats medels with cutaneous chronic wounds, we treat different methods with different groups as written before, and choose 5 rats from every groups randomly 2 and 7 days after the treatment, execute them and cut the granulation tissues,10 woulds for every group. Data that needed to be analized the inflammation, new-born blood vessels,area of healing and crust by HE stain and immunostaining.Results:1. Induced the models of type 2 diabetes mellitus by 2 month feeding with high glucose and grease and streptozocin injection is very successfully and stably.2. After passaged, BMSCs were grown quickly with long-fusiforn shape. VEGF had been transfected into BMSCs, and stable expressed. The transfected BMSCs grew well in the surface of collagen sponge.3. We can see BMSCs grown well on the surface of skin healing and migrated to granulation tissue.4. The macroscopy and rate of wounds healing showed:DM-MSCs groups and DM-MSCs-VEGF groups have the same recovery rate, which is obviously better than the rate of DM-blank groups and DM-collagen groups; DM-collagen groups have the better rate than DM-blank groups. HE stain showed:the wounds of DM-MSCs groups and DM-MSCs-VEGF groups healed better than DM-blank groups and DM-collagen groups. The immunohistochemistry stain suggested:the expression of a-SMA and PCNA in DM-MSCs groups and DM-MSCs-VEGF groups was higher than DM-blank groups and DM-collagen groups at 7 days; compare with DM-blank groups and DM-collagen groups,the color were more obvious in DM-MSCs groups and DM-MSCs-VEGF groups by TNF-a stain at 2 days and VEGF stain at 7 days.Conclusion:1,The rat type 2 diabetes mellitus model could be induced by 2 month feeding with high glucose and grease and streptozocin injection.2,VEGF had been transfected into BMSCs, and stable expressed. BMSCs and the transfected BMSCs grew well in collagen sponge, and tissue-engineered skin was constructed successfully. 3,BMSCs can survive and migrate in the skin wound.4,The tissue-engineered skin could improve the skin healing in T2DM. And the better repairing could be obtained as VEGF.