The Research On Rat Bone Marrow Mesenchymal Stem Cells Differentiating Into Epidermal Cells And Repairing Skin Wound Defect

Objective: Investigate the ability of bone marrow mesenchymal stem cells(MSCs) differentiating into epidermal cells in vitro under certain condition and experimental study on healing rat skin wounds and rebuilding epidermis by transplantation with MSCs only or MSCs with necessary inductor so as to provide experimental document for treatment of severe burns by transplantation with bone marrow mesenchymal stem cells from bone marrow .Methods:1. MSCs were isolated from healthy rat and purifided by density gradient centrifugation, collect the Mononuclear cells(MNCs) layer. All cells were seeded into six-pore plaque containing Dulbecco's-modified Eagle's Medium-Low Glucose (DMEM-LG), penicillin 100 U/ml, streptomycin 100μg/ml, and 10% fetal calf serum (FCS). MSC cultures grew at 37°C in 5% CO2. Nonadherent cells were removed after 24 hours. The medium was changed subsequently every 2 days. Two weeks later the culture reached 80% confluency. MSCs were recovered using 0.25% Trypsin-EDTA and replated at a density of 5,000–6,000 cells per cm2 of surface area as passage 1 (P1) cells .When the amount of MSCs reached 1×106cells ,special antigen of CD34,CD29,CD90 and CD45 was tested by flow cytometry.2. Confluent cells(MSCs)from passages P4 were cultivated in six-pore plaque containing DMEM-F12, 20% burns rat serum.Medium was changed every 3 days. When culture reached 80% confluency, 0.25% Trypsin-EDTA was used to recover and replate them. Epidermal cells (cytokeratin +) was tested with immunohistochemical methods and FCM(flow cytometry)after 14 days.3. The animal transplantation experiment : Preparing animal model and MSCs which are induced and are not induced are replanted. Select 24 healthy Wistar rats, whose average weight are 200g, are half male and half female, and are grouped into three groups, each group are eight rats, namely treatment group A (induction group), treatment group B ( without inducing group) and control group C ( no cell group); The rats'back linea median, each manufacture diameter is about 5cm circular entire thick skin damage injured area, immediately byⅠcollagen is a carrier, the BrdU mark, MSCs induced and MSCs without inducing are spread respectively onto injured area in the treat group A and B, but the control group C only are spread byⅠcollagen in the injured area.On injured areas of each group, spread the vaseline gauze, after then suture gauze and injured area, raise them respectively .4. Injured area observation: According to formula,note area of regenerate skins wound on aseptic transparent membrane at fourteenth day after surgery respectively, compare heal time of groupA or B with heal time of groupC and compare shrinkage rate of groupA or B with shrinkage rate of groupC.Wound'shrinkage rate ( %) = regenerate skins wound'area / primary skins wound'area×100%5. Pathology and immunohistochemistry stain test: Select experimental rats after four weeks,cut off regenerate skins which diameter are 1cm at rats'back center,make specimen slices,stain them with H-E and watch cellular form. And serial section, use theSABC immunohistochemistry stain test. Strictly according to BOSHIDE Corporation reagent box instruction operate, dye with diaminobenzidine (DAB). Mouse anti-BrdU monoclonal antibody (in Beijing ZhongShan JinQiao Biology Company) work density is 1∶100, goat anti-mouse multiclonal antibody work density is 1∶100. Observes the dyeing result with the microscope, the cell nucleus assumes the yellowish brown color is the BrdU positive cell.Results:1. Phase contrast microscopy from cells after cultivating 4 days demonstrated a fibroblast-like, spindle-shaped morphology. In later 14 days, the spindle-shaped cells began to display a broadened, flat morphology, and arrange like a group of fish. FCM(flow cytometry) analysis showed that 96.87±2.54% cells expressed MSCs typical antigens CD29,2.65±0.56% cells expressed CD34 which is a antigens of Hemopoietic stem cell. 90.37±3.55% cells expressed CD90 and 1.62±0.16% cells expressed CD45.2. Epidermal cells'identification: MSCs were induced by 20% burns rat serum for 14 days , FCM was used to test CK positive cell ,namely epidermal cells'express rate were 7.22%,but comparison were 0.23%.In the meantime, collected cells and used immunohistochemistry stain to test CK positive cell of plasma becoming yellowish brown which were epidermal cells.3. The injured area observation: In two groups injured areas were both dry, the area reduces day after day, not infective sign. At fourteenth day after surgery,in treatment groupA the injured area's average shrinkage rate was78.2%±9.8%, in treatment group B the injured area's average shrinkage rate was79.1%±10.4%,in the control group C the injured area's average shrinkage rate was 70.7%±10.2%, two groups'disparity (A group and C group, B group and C group) was significant (P<0.05)especially, but between treatment groups'disparity (A group and B group) was not significant (P > 0.05).In treatment group A the injured area's average heal time was 15.4%±0.9d, In treatment groupB was16.0%±1.2d, in the control group C the injured area's average heal time was 19.5%±1.0d, two groups'disparity (A group and C group, Bgroup and C group) was significant (P<0.05)especially,but between treatment groups'disparity (A group and B group) was not significant (P > 0.05)。4. Compared skin regeneration when MSCs alone were transplanted with skin regeneration when MSCs with the induction factor combination were transplanted: (1) Conventional pathology observation: after four weeks,respectively observed skin regeneration in the pathological section of three group,in treatment group AorB regenerated skin appendix were obviously rich, the number of epidermal cells obviously increased, the levels of cells increased, cuticular layer obviously thickened. (2) Immunity histochemistry dyeing result: after four weeks, in the skin tissue slice of treatment groupAorB BrdU dyeing positive cell could be found, the positive cells were located at the skin appendix hair-follicle inner layer epidermis root sheath and the regenerated epidermis stratum basale, the thorn level, this kind of cells in the morphology presented square shape or polygon, not obvious difference with periphery epidermal cells and its neighboring growth.Conclusion:1. Because MSCs which are induced by burns rat serum are able to differentiate into epidermal cells,make it clear that MSCs have ablility of differentiation into ectoblast histiocyte under certain condition.2. compares MSCs alone to transplant the group and with the essential inductor combination transplant group, his/her the skin injured area in the average shrinkage, the average heals in the time not to have statistical the difference.3.When MSCs or MSCs with necessary inductor are transplanted, heal or regeneration of rat skin wounds are obviously better in groupA or B than heal or regeneration of those in groupC,not only grow speed is quicker,but regeneration of skin accessories ,for example hair follicle ,can obviously improve, to provide experimental document for clinical transplantation treatment of a large area of severe burns(is skins which are used for transplantation can get easily and promot skin wound defect regeneration quicker,in the end ,own normol skin'function.)...