关键词: 皮肤缺损, 内皮祖细胞, 外泌体, 血管内皮生长因子A

Abstract: Objective: To explore the effects of exosomes derived from bone-marrow endothelial progenitor cells(EPCs-Exos)on the angiogenesis and collagen deposition in vitro,and to illustrate the possible mechanism for EPCs-Exostoaccelerate the cutaneous deficiency repair.Methods: The endothelial progenitor cells (EPCs) were isolated, cultivated and identified; at the same time, the EPCs-Exos were also isolated and identified. The EPCs-Exos uptake in EPCswas observed and analyzed.16 rats were randomly divided into 4 groups, and then the models of skin defect were established, respectively. The equal volume of PBS and 50, 100, 150 mg·L^-1 EPCs-Exos were injected into the area around skin defect of the rats in 4 groups. The wound closed sizes on the 0, 3rd, 7th and 14th days were measured, and Masson's trichrome staining and CD31 immunofluorescence staining were performed on the 14th day for evaluating the tissue healing efficacy after EPCs-Exos treatment. In vitro,the mediums containing PBS and 50, 100, 150 mg·L^-1 EPCs-Exos were used to culture the human umbilical vein endothelial cells (HUVECs), respectively. Scratch test and tubule formation assay were used to detect the migration and capillary network formation of HUVECs. At the same time, Western blotting was used for analyzing the expression level of angiogenesis related gene vascular endothelial growth factor A (VEGFA) in HUVECs.Results: The primary EPCs were isolated and identified successfully, and EPCs-Exos were purified and characterized.The CD31 immunofluorescence staining and double staining of DiL-ac-LDL and FITC-UEA-I of EPCs were positive. The electron microscope results showed that EPCs-Exos were nearly spheroidal, with the diameter about 40-100 nm. For the models of rat skin injury treated by EPCs-Exos, with the increasing of injection doses, the sizes of skin defect scar were gradually reduced, the degrees of scar healings were gradually increased,and the differences between various groups were statistically significant (P< 0.05). EPCs-Exos promoted the collagen maturity of healing skin in a dose-dependent manner; on the 14th day, the effect in 150 mg·L^-1 EPCs-Exos group was the most significant. In vitro, EPCs-Exos promoted the migration and capillary network formation of HUVECs and increased the expression level of VEGFA; the migration rate,the net number of branches and the expression level of VEGFA in 150 mg·L^-1 EPCs-Exos group were significantly higher than those in 50 mg·L^-1 EPCs-Exos group and PBS group (P< 0.05).Conclusion: EPCs-Exos can promote the repair of traumatic skin defect of the rats by positively regulating the vascular endothelial cell function.

Key words: endothelial progenitor cells, exosomes, cutaneous deficiency, vascular endothelial growth factor A