| Objective:1. To establish the rabbit type 2 diabetes mellitus models and the skin defect model with type 2 diabetes mellitus, then some data of histology and immunohistochemistry was detected to approach the factors related to the skin healing with type 2 diabetes mellitus.2. Basic fibroblast growth factor (bFGF) gene was transfected into bone marrow mesenchymal stem cells (BMSCs), and combined the cells with SIS 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:The hole experiment was devided into 3 parts:Part one: The study of healing factors of skin defect with type 2 diabetes. The rabbit type 2 diabetes mellitus models was induced by 2 month feeding with high glucose and grease and streptozocin injection. The blood sugar, blood fat and insulin was detected at the times of 2 month feeding, 2 weeks , 4 weeks, and 8 weeks after the injection. Then the skin defect was estabolished based on the type 2 diabetes model, and the defect skin healing was compared at the 3 days, 1 week, 2 weeks, 4 weeks with control group, included the inflammation, area of healing and crust. Also the HE stain and immunohistochemistry stain was taken to compared the expression of PCNA, CD8, MMP9 and collagen fibers.Part two: Construction of tissue-engineered skin using basic fibroblast growth factor gene transfected bone marrow mesenchymal stem cells combined with small intestinal submucosa. BMSCs were obtained from Japanese big-eared rabbits, and in vitro cultured. Then the subcultured BMSCs were transfected by pCDNA3.1 plasmid, followed by incubation on swine SIS to construct the tissue-engineered skin. The growth of cells and phenotype of BMSCs were detected by flow cytometry. In addition, the result of transfecting BMSCs with pCDNA-bFGF vector was measured by Western blot, and the structure of tissue-engineered skin was observed.Part three: The tissue-engineered skin constructed in part 2 was transplanted to the skin defect with type 2 diabetes, and the control group was also set up. Then the defect skin healing was compared at the 3 days, 1 week, 2 weeks, 4 weeks with control group, included the inflammation, area of healing and crust. Also the HE stain and immunohistochemistry stain was taken to compared the expression of PCNA, CD8, MMP9 and collagen fibers.Results:Part one: The rate of models of type 2 diabetes mellitus induced by 2 month feeding with high glucose and grease and streptozocin injection was 70%. In the models of skin healing, the healing of control group was faster than the DM group, as same as the area of healing(P<0.05). HE stain suggested: the emergence time of granulation tissue was later and less. The neogenesis of vessels in granulation tissue was not active. The during of Inflammatory reaction was longer, and severe Inflammatory reaction and necrosis could be deteced in advanced stage. The immunohistochemistry stain showed: the expression of PCNA in DM group was higher than control group at 3 days and 1 week after surgery(P<0.05), but at 2 and 4 weeks there were no Significant difference between the 2 groups; the expression of CD8 in DM group was higher at 3days, 1 week and 4 weeks, but lower at 2 weeks(P<0.05); the expression of MMP9 in DM group was higher at all time, as some as the Van Geisen stain.Part two: After passaged, BMSCs were grown quickly with long-fusiform shape. The cells were positive expressed CD90 and CD44, but negative expressed CD45. bFGF had been transfected into BMSCs, and stable expressed. The transfected BMSCs grew well in SIS.Part three: The macroscopy and HE stain showed: there were no significant difference between the tissue-engineered skin group and control group at the 3 days, but showed the significant difference between two groups at 1,2 and 4 weeks(P<0.05). The immunohistochemistry stain suggested: the expression of PCNA in tissue-engineered skin group was higher than control group at 3 days, 1 and 2 weeks, but lower at 4 weeks(P<0.05); there were no significant difference between the two groups in the expression of CD8; there were no significant difference between the two groups in the expression of MMP9 at 3 days and 1 weeks, but significant difference at 2 and 4 weeks(P<0.05); the color were more obvious in tissue-engineered skin group by Van Geisen stain at all time; the HCK stain werepositive at 4 weeks.Conclusion:Part one: 1. The rabbit type 2 diabetes mellitus model could be induced by 2 monthfeeding with high glucose and grease and streptozocin injection. 2. The capability ofskin healing in diabetes mellitus was poor, the slow union and disunion occurrence. 3. Theemergence time of granulation tissue was later and less; the neogenesis of vessels in granulationtissue was not active; the capability of hyperblastosis and repairing was hysteresis and disorder;the ECM disintegration was excess and local immunity was abnormal, excess and disorder; andthe main reason of poor capability in skin healing with DM might be the severe and long duringinflammatory disorder.Part two: 1. BMSCs were grown quickly with long-fusiform shape. 2. bFGF had been transfectedinto BMSCs, and stable expressed. 3. The transfected BMSCs grew well in SIS, andtissue-engineered skin was constructed successfully.Part three: 1. The BMSCs could differentiate into epidermis cells.2. The tissue-engineeredskin could improve the skin healing in DM. 3. The better repairing could be obtained as bFGF.
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