The Effect Of Serum-derived Exosomes On The Wound Healing In Scalded Mice

Objective:1.To confirm whether the serum-derived exosomes promote the healing of scalded wound.2.To investigate the underlying mechanism of serum-derived exosomes promoting the healing of scald wound.Methods:1.Electron microscope was used to identify the morphology of the exosomes extracted from rat serum by Exo Quick reagent.The expression of exosomal marker protein CD63 and HSP70 was detected by western blotting.Using NTA to detect the diameter of exosome.2.NIH mice were selected to establish the skin deep two degree scald model of skin.The HE staining was used to judge whether the model was successful.3.Serum-derived exosomes were injected subcutaneously into the scalded skin.The scalded areas of each group were regularly measured,and the scalded skin was removed and stained with HE.The pathological changes of skin tissue were observed by microscope,and the curative effect was evaluated.4.Fluorescence microscope was used to observe whether the exosome could enter into the skin tissue and cells.5.MTT assay was used to detect the effect of serum-derived exosomes on the proliferation of mouse fibroblast.6.Flow cytometry was used to detect the effect of serum-derived exosomes on apoptosis of Hacat cells.7.The effect of serum-derived exosomes on the migration of Hacat cells was evaluated by cell wound scratch assay.8.Western blotting was used to detect the AKT phosphorylation level in the scalded skin treated with or without serum-derived exosomes.9.Cell immunofluorescence was used to detect the effect of serum-derived exosomes on content of VEGF protein in vascular endothelial cells.10.RT-q PCR was used to detect the effect of serum-derived exosome on VEGF m RNA in vascular endothelial cells.Results:1.Serum-derived exosomes isolated by Exo Quick reagent exerted a saucer-like bilayer membrane structure.The exosomal marker proteins CD63 and HSP70 were detected in exosomes.The uniformity of exosome particle size was good,were all about 127 nm.2.The deep two degree scald model of NIH mice skin was successful established.The healing rate of exosome group was higher than that of control group.HE staining showed that serum-derived exosomes could effectively repair the accessory structure of skin and improve the arrangement of collagenous fibers.3.Compared with the control group,serum-derived exosmes promoted the proliferation of mouse fibroblasts and promoted the migration of Hacat cells,as well as reduce the apoptosis of Hacat cells.4.Compared with the control group,serum-derived exosmes reversed scald-induced AKT dephosphorylation in skin tissues.5.Compared with the control group,serum-derived exosmes increased the content of VEGF protein in vascular endothelial EA.hy926 cells.6.Compared with the control group,serum-derived exosomes decreased the level of VEGF m RNA in vascular endothelial EA.hy926 cells.Conclusion:1.Serum-derived exosomes could promote the healing of skin in scalded mice.2.Serum-derived exosomes may promote cell proliferation and migration by inducing AKT phosphorylation,and at the same time exert their role in promoting wound healing by angiogenesis of VEGF protein.