ObjectiveThe biological characteristics of VEGF and hMSC will be involved in this experiment. Utilizing the Wistar rat to establish ultra-ratio random flap, observe the reestablishment of blood circulation in ischemic region of different division of animal skin flap in short term, by trying the local application of the VEGF, hMSCs, and the culture of VEGF and hMSC. Investigate the effect that the combination of VEGF and hMSCs make to the solution of the distal pedicle ischemia of ultra-ratio random flap, the enhancement of the skin flap survival rate, and the quality restoration of wound tissues.Methods1. Isolation, culturing and identification of human bone marrow mesenchymal stem cells:Isolate and culture bone marrow mesenchymal stem cells by both adherence separation and density gradient centrifugation. The cells which are sub-cultured for more than three generations can be used in the follow-up animal experiment. Immunohistochemistry and flow cytometry (FACS) united identification will be adopted. Besides, according to MSCs has the characteristic of multiplex differentiation potential, use reduction to absurdity.2. Establishment of animal model:Use rat back as the operation area, designing the skin flap approach after routine disinfection. Mark out the 2×5cm rectangular flap by gentian violet. Strengthen infiltration anesthesia in local area by 0.5% lidocaine. Incise full-thickness skin along the designed line down to the superficial layer of muscular fasciae by the 11th scissors slice. Use the dissector peel off the clearance on the muscular fasciae, make the distal pedicle of the skin flap completely free, and then establish the random flap model. Use lukewarm physiological saline gauze to stop bleeding by compression in local bleeding point. Stitch up the wound by the technique of interrupted apposition sutures along number 0 line. Besmear with aureomycin eye ointment, inject Penicillin 200000RU, and inject RECOVERY-4 in a ratio of 1.5:1 till the end of suturing. Put the rat into the isolated cages after it can move about freely.According to the need of experiment designing, divide the experimental animals randomly into four groups, 10 each group. Marked with A, B, C, D, which are blank control group, VEGF group, hMSCs group, VEGF+hMSCs group.After blank control group set up, suture the flaps in situ. VEGF group set up in the flaps then inject 300ng VEGF to six different injection sites in the fascia, suture in situ; hMSCs group set up in the flaps then smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells).VEGF + hMSCs group set up in the flaps, inject 300ng VEGF to six different injection sites in the fascia, and smear the wound with cultured bone marrow mesenchymal stem cell cultures (containing about hMSCs1×107 cells),suture in situ.3. Make continuous clinical observation and record the back flap of the rats during 14 days after operation. Observe whether there is immunological rejection , such as diarrhea, Camponotus, Alice hair, depression and skin ulcers to the experimental animals. Observe the flap color, thickness, elasticity, exudation, hair growth, necrosis area and acupuncture bleeding situation, then record in detail. on the fourteenth day after operation, use sulfuric acid paper describe the area of ischemic necrosis tissue on the distal end of the flaps, then add the number of grids in the grid paper to calculate the survival area and converte to percentage survival, that is the average survival area rate of flap. Data on the results obtained were statistically analyzed.4. Sacrificing the rats on the fourteenth day after operation, cutting the flap 4cm apart the distal pedicle of each rats, detecting the flaps with the histological examination and immunohistochemical methods. Observing the volume and density of microvessels of the distal pedicle under the microscope. Data on the results obtained were statistically analyzed.Results1. The bone marrow-derived mesenchymal stem cells cultured isolated are typical spindle-shaped fibroblast-like. 16th-30th days during isolated culture, the cell confluence is above 80%-90%, which arranges in imbricate or Vortex-like shape. detecting surface marker with Flow Cytometry, we found it didn’t express surface markers of hematopoietic cells, which means CD34、CD45、HLA-DR sustained negative, CD29、CD44、CD73、CD105、CD166 sustained positive. This stem cells can be induced to fat and cartilage differentiation, which proves bone marrow-derived mesenchymal stem cells have multi-directional differentiation potential.2. There is immunological rejection in each experimental group during 14 days after operation. On the fourteenth day after operation, the survival rate of flap: VEGF+hMSCs group > VEGF group> hMSCs group> blank control group.3. HE stained histological examination on the fourteenth day after operation, we found in group D, there are the most abundant granulation tissues, cell components and new capillaries, fibroblasts increased; in B group and C group, there are also more abundant granulation tissues, cell components and new capillaries; in A group we could see a large number of inflammatory cell infiltration, necrotic tissues, less cell components and new capillaries.4. The fourteenth day after operation, the results of the CD34 factor polyclonal antibody immunohistochemical staining showed that the vascular endothelial cells express factor CD34 positive, and the cytoplasm stained brown. in each group under the microscope we could observe the new relationship between the number of microvessels and microvessel density (MVD): VEGF + hMSCs Group> VEGF group> hMSCs group> control group.DisscutionThe separately local application of VEGF and hMSCs could increase the survival rate of the flap. The effects of the united application of VEGF and hMSCs will increase the overwhelming survival rate of the flap is much superior to the separate application of VEGF and hMSCs. The united application of the culture of VEGF and hMSCs may rebuild the ischemic region and blood circulation in the short term, and solve the distal pedicle ischemia of the ultra-ratio flap, increasing the reparation quality of the survival rate of the flap and epidermis organization.