| Objective: Tension is an important factor affecting skin wound healing.Larger wounds have greater mechanical tension,and high tension often leads to delayed wound healing and ultimately leads to the formation of pathological scars.The proliferation,migration and differentiation of skin fibroblasts are considered to be the key factors in the process of wound healing.Our previous studies confirmed that high amplitude tension will stimulate fibroblast proliferation and differentiation and induce myofibroblast formation.Adipose-derived mesenchymal stem cells(ADMSCs)have a positive effect on wound healing,but there is a potential carcinogenic risk.In recent years,the application concept of stem cells has been extended to use stem cell-derived exosomes to affect the functions of target cells and regulate the body's microenvironment.At present,the effect of ADMSCs-exos on the healing of tension wounds is unclear.This study explored the mechanism of ADMSCs-exos to promote the healing of tension wounds,provide a theoretical basis for improving the repair efficiency of tension wounds and preventing hypertrophic scar formation,and provide new ideas for clinical treatment of tension wounds.Methods:1.NSFB and ADMSCs were isolated from healthy skin and adipose tissues.Exosomes were purified from human ADMSCs by differential ultracentrifugation.BCA method was used to detect the exosome concentration.the morphological characteristics of exosome particles was observed by Transmission Electron Microscopy(TEM),the diameter distribution of exosomes was measured by Nanoparticle Tracking Analysis(NTA)and Western blot assay was applied to detect the characteristic cell surface marker derived from exosomes.2.In vitro experiments,after treating normal skin fibroblasts(NSFB)with exosomes of different concentrations(0ug / ml,25 ug / ml,50 ug / ml and 100 ug / ml),the CCK-8method and cell scratch assay was used to examined the effects on the proliferation and migration of fibroblasts.After the exosomes treatment,Western blot assay and qPCR method were used to measure the mRNA synthesis and the protein expression of the collagen type I(Col – I),the collagen type III(Col-?)and the ?-smooth muscle actin(?-SMA).At the same time,Western blot was used to detect the protein expression of Wnt2 b and ?-catenin to determine the activation of Wnt / ?-catenin signaling pathway.3.A full-skin thickness defect model on back of SD rats was constructed,and a certain tension was applied to the wound circumference by suture and fixing the rubber ring to establish a tension wound model.Rats were randomly divided into Exo group,PBS group and blank control group.The 100 ug exosome suspension and PBS buffer both with a final volume of 200 ul were given by local injection.The blank control group give no treated.Observe the wounds on 0d,3d,7d and 14 days,and take photos to analyze the healing.On the 14 th day,the mice were sacrificed,and specimens were cut at 2 mm around the wound surface or scar tissue edge,and subjected to IHC test for detecting Wnt2 b and?-catenin protein expression.Results:1.The morphology of ADMSCs-exos observed under electron microscope was cup-shaped,with bilateral membrane structure.The concentration of exosomes obtained from 320 ml of cell supernatant measured by BCA method was about 1.0mg / ml,and the particle diameter was distributed between 40-100nm;Western blot assay successfully detected surface specific protein markers CD9 and CD63.2.The results of the CCK-8 test showed that after 24 h and 48h(P <0.05)of ADMSCs-exos,the proliferation capacity of fibroblasts was significantly increased and showed a concentration-dependent.The scratch test results showed that the migration rate of fibroblasts increased with the increase of exosome concentration,and the difference in residual scratch area analysis between ADMSCs-exos group and blank group at 24 h and48h(P <0.05),the difference was statistically significant.3.In the rat tension wounds,the general appearance of the wounds showed that after7-day and 14-day ADMSCs-exos treatment,the residual area of ??the wound in the Exos group was significantly lower than that in the PBS group and blank control group.4.The results of Western blot and qPCR showed that the protein and mRNA expressions of Col-I and Col-? on fibroblasts under the effect of ADMSCs-exos were significantly increased,while the expression of ?-SMA was significantly reduced,the difference was statistically significant.However,when the concentration of ADMSCs-exos increased to100ug/ml,the opposite effect on fibroblasts was observed.The SD rats IHC results showed that the Exos group also showed high expression of collagen.5.Westernblot assay showed that the protein expression of Wnt2 b and ?-catenin after ADMSCs-exos treatment was significantly increased than that in PBS group and blankcontrol group.IHC results of Rats tissue showed similar results.The protein expression level of Wnt2 b and ?-catenin in Exos group was higher than that in other two groups.Conclusion:1.Successfully obtained exosomes derived from human adipose mesenchymal stem cells(ADMSCs-exos)by ultracentrifugation and successfully identified them.2.ADMSCs-exos can enhance the activity of fibroblasts in vitro by promoting the proliferation,migration and collagen expression of fibroblasts.3.ADMSCs-exos plays an important role in promoting wound healing in tension wounds,which may play a positive role on wounds by activating Wnt / ?-catenin signaling pathway. |
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