Effects Of Exosomes Derived From Vascular Endothelial Cells On Wound Healing

Background:Wound healing is a complex process,coordinated and controlled by various cells,among which vascular endothelial cells(VECs)play a crucial role.Wound formation activates VECs to build neovascular to promote wound healing,under the regulation of different factors.Once the dynamic balance of this process was broken,delaying wound healing for more than one month,the chronic wound will appear.Exosomes derived from VECs(VECs-exos)are microvesicles secreted by VECs through autocrine or paracrine,carrying substances such as RNA and proteins,which mediate critical signal transduction during wound healing.Nowadays,wound healing researches in the world mostly used VECs as target cells,but how VECs-exos affect wound healing still remains unclear.Objective:To investigate the effect of VECs-exos on normal wound healing and diabetic chronic wound healing via experiment in vitro and vivo.Methods:1.Extraction and identification of exosomes from HUVECs.We firstly isolated and cultured HUVECs to select the 3rd generation cells for flow cytometry identification,and simultaneously collected the culture medium used during the 1st-6th generation cell culturation to extract exosomes by overspeed centrifugation.Then we used transmission electron microscope(TEM)to observe the morphology of exosomes and nanometer particle size meter(NANOPHOX)to measure the diameter distribution of exosomes.Besides,surface makers of exosomes were detected via Western Blotting and the protein concentration was determined by BCA method.2.To investigate the effects of exosomes derived from HUVECs on the proliferation and migration of Fbs and HUVECs and the angiogenesis ability of HUVECs under normal and high glucose conditions respectively.Fbs and HUVECs were isolated and cultured to the 3rd generation,and then exposed to 25mmol/L D-glucose(simulated hyperglycemia,HG group)and 5mmol/L D-glucose(simulated normal glucose,NG group)for 24 h.After that,we used CCK-8 kit to detect the proliferation of HUVECs and Fbs intervened by different concentrations of exosomes both in HG group and NG group.Next,the migration ability of HUVECs and Fbs intervented by exosomes and equivalent amounts of PBS,both in HG group and NG group,were detected by Scratch and Transwell assays.Finally,angiogenesis assay was used to determine the angiogenesis ability of HUVECs under the same conditions.3.To investigate the effects of exosomes derived from HUVECs on the wound healing of normal and diabetic rats.Diabetic rat model(random blood glucose was higher than 15mmol/L)was induced by high fat and sugar diet combined with streptozotocin injection(35mg/kg,injection: 2 times,with a 24 h interval).And 20 normal rats(HG group)and 20 diabetic rats(NG group)were involved in the experiment.We established a circular wound model on the back of rats with 1.5cm in diameter,and divided the HG group and NG group into experimental group(injected with exosomes)and control group(injected with equivalent amount of PBS)randomly.And finally,the wounds were observed on day 0,7 and 14 and samples of the wounds were taken on day 7 and 14 for Masson staining and CD31 staining.Results:1.Exosomes secreted by HUVECs were elliptical structure with complete membrane with diameters of 71.68±17.72 nm under the TEM,expressing CD63,and the protein concentration which was about 2.88 ug /ul.2.The exosomes derived from HUVECs could promote the proliferation of HUVECs and Fbs in HG group and NG group,and had the strongest ability at the concentration of0.72ug/ul.The migration ability of HUVECs and FBs in HG group and NG group could be enhanced under the action of exosomes derived from HUVECs.And exosomes derived from HUVECs could also promote the angiogenesis ability of HUVECs under both normal and high glucose conditions.3.The residual wound area of experimental groups was smaller and the healing rate of experimental groups became faster than that of control groups from the 7th day both in HG group and NG group.On day 7 and 14,the collagen of experimental groups packed much tighter showed by Masson staining and the positive expression of CD31 was stronger in experimental groups.Conclusion:VECs-exos can promote the proliferation and migration of Fbs and HUVECs under both normal and high glucose conditions,and ultimately promote the healing of normal and diabetic chronic wounds.