Effects Of TNF-α-induced Human Umbilical Cord Mesenchymal Stem Cells Exosomes On Repair Cells And Wound Healing

Objective:Mesenchymal stromal cells derived Exosomes(MSC-Exos)can effectively promote wound healing.However,the micro environmental conditions of cell culture and cell transplantation pathological inflammation can greatly affect the paracrine effect of Mesenchymal Stromal Cells(MSCs)and the biological effect of MSC-Exos.Inflammation pretreatment can regulate immune functions and repair functions of mesenchymal stem cells.This study compares the difference between human umbilical cord mesenchymal stem cells(HUCMSCs)derived exosomes treated with tumor necrosis factor alpha(TNF-Exos)and cultured under normal condition(Nor-Exos)to study the function of TNF-Exos in promoting wound healing,and use the vascular endothelial cells and fibroblasts to research the repair mechanism.。Methods:(1)Isolation and identification of normal HUCMSC-Exos and TNF-αpretreated HUCMSC-Exos:human umbilical cord mesenchymal stem cells were extracted by tissue attachment method and identified by flow cytometry(FCM).Human umbilical cord mesenchymal stem cell derived exosomes(HUCMSC-Exos)were extracted by differential centrifugal method.Morphologies of HUCMSC-Exos were observed by transmission electron microscopy.Particle size of HUCMSC-Exos was identified by nano particle tracking analyzer.Western Blot(WB)was used to identify the expression of HUCMSC-Exos specific labeled proteins CD9 and CD63.Based on relevant literature,HUCMSCs was stimulated with 20ng/m L TNF-αfor 48h,and exosomes were extracted with the same method.(2)Effects of different HUCMSC-Exos in the model of full-thickness skin wound defect in mice:the diameter of the wound was 1cm~2.Nor-Exos or TNF-Exos were injected with 200μg,around the wound surface to observe and calculate the wound healing rate.(3)Effects of different HUCMSC-Exos act on vascular endothelial cells:human umbilical vein vascular endothelial cells were stimulated by Nor-Exos or TNF-Exos at concentrations of 10μg/m L,50μg/m L,100μg/m L,respectively.The effect of different exosomes on wound healing was observed in vitro by tubular experiment.(4)Effect of different HUCMSC-Exos act on human skin fibroblasts(HFF):With the Nor-Exos or TNF-Exos concentration of 100μg/m L acting on human skin fibroblasts,we study the different effect of exosomes on proliferation,migration,inflammation,and repair the relevant factor m RNA expression of HFF,by using the method CCK8,cell scratch experiment,real-time quantitative PCR(RT-q PCR)experiment.Results:TNF-αstimulated HUCMSC-derived exosomes significantly promoted wound healing.The effect was better than that of HUCMSC-derived exosomes cultured under normal conditions(P<0.05).In vitro studies showed that exosomes had dose-dependent effect.When the concentration of exosomes is 100μg/m L,it shows the best result.TNF-αpretreated HUCMSC-Exos could significantly promote the angiogenesis of human umbilical vein endothelial cells in vitro(P<0.05),but there was no obvious difference between Nor-Exos(P>0.05).TNF-αpretreated HUCMSC-Exos could significantly promote the proliferation and migration of human skin fibroblasts in vitro,reduce the expression of inflammatory factors,down-regulate the expression of pro-fibrosis factors to inhibit the formation of scar(P<0.05),but no obvious difference had been found between the Nor-Exos(P>0.05).Conclusion:(1)Compared with HUCMSC-Exos cultured under normal conditions,HUCMSC-Exos pretreated with TNF-αcould significantly promote the wound healing in the model of full-thickness skin defects in mice.(2)Nor-Exosomes and TNF-αpretreated HUCMSC-Exos can promote wound healing and reduce scar formation by promoting vascular endothelial cell tube formation and promoting the migration and proliferation of human skin fibroblasts and reducing the m RNA expression of TGF-β,IL-6,Collagen I andα-SMA.(3)HUCMSC-Exos pretreated by TNF-αcan better promote wound healing.The main target of HUCMSC-Exos pretreated by TNF-αis not in vascular endothelial cells and fibroblasts,which may affect wound healing by changing inflammatory microenvironment and inflammatory cells or inflammatory repair cells such as macrophages in wound.