| Objective: To investigate the feasibility of local frostbites on SD rats; To explorethe contribution of adipose derived stem cells (ASCs) to frostbite wound healing andprovide a new idea for the mechanism study of frostbite wound healing and clinicaltreatment in the future．Methods: Adipose derived stem cells (ASCs) of SD rats were harvested andcultured in vitro routinely and the three passage was identified by flowcytometry．Different final concentration of5-Bromo-2-deoxyUridine(5-BrdU) was taggedASCs, and did cell immunofluorescence staining observing the markers by the help offluorescent microscope, and the next step was building up SD rats frostbite models: todesign a round wound of22mm in diameter in the back of the SD rats, and, by manualpressure pump system at a constant speed, pump and spray the liquid of nitrogen ontothe experimental area, keeping the Injury time for0,3seconds,5and8secondsrespectively. Making statistics analysis by the application of laser Doppler perfusiondetection the following day, Then there was the HE staining of the skin tissue of thosethat had been killed. In the following three days, a week, up to2weeks after thefreezing, a general observation was made to the32wounds of the8SD rats. Applying1ml of the third generation of Brdu-labeled stem cells (concentration of1x10(6)/ml) tothe frostbitten SD rats. Getting the rats in5groups: Group A, a blank control group,Group B injected with Brdu-labeled ASCs via the tail vein, Group C, injected with aninjection of1ml of0.9sodium chloride via tail vein and Group D, a local injections ofBrdu-labeled ASCs cells, Group E, local injection of1ml0.9%sodium chlorideinjection, all killed7to14days respectively days after transplantation, cutting frostbitewound tissue for fixation, paraffin embedding, sectioning, HE staining and immune fluorescence staining. Examinations were made to investigate transplanted cells aroundthe wound site and identify the contribution of ASCs to frostbite injury repair indifferent time during wound healing．Results: Flow cytometry showed that ASCs pressed CD29，and CD44，but noCD31、CD45．The work of manul liquid nitrogen pump can result in severe frostbitewounds of SD rat skin on his back in8seconds. The incubation rate of stem cells of thethird generation of fat source of stem cells in vitro marked10mmol/lBrdU was about80%or so in48hours. The experimental group of5-Brdu tracer in vivo: positive5-Brdu labeled cells are visible, gathered locally or along the edge of the wound of therats,14days after cell transplantation; In the control group there’s no obviousaggregation phenomenon found; results of resistance to5-BrdU/FVIII and resistance to5-BrdU/vimentin immune double label technique test show that, in the wound areas,there’s certain differentiation of the ASCs into vascular endothelial cells and fibroblastsin wound healing.Conclusion: ASCs, with stem cell properties, is convenient in finding its sources,speedy in the culture in vitro and amplification. Frostbite in8seconds with theapplication system of liquid nitrogen pump manually can be used as a standard toconstruct severe frostbite models. Autologous adipose derived stem cells of SD rats arefeasible in frostbite wound healing. |
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