Effect Of Umbilical Cord Mesenchymal Stem Cell Exosomes On Acute Skin Wound Healing

Objective To investigate the effects of exosomes of umbilical cord mesenchymal stem cells（uc MSC-exos）on proliferation and migration of human skin fibroblast（HSF）;preliminary study on the effect of uc MSC-exos on acute skin wound healing in mice and its related mechanismMethods The exosomes were extracted by ultracentrifugation.The exosomes were identified by electron microscopy,Western blot and particle size analysis.Firstly,3～5 generations of skin fibroblasts（human skin fibroblast,HSF）were cultured in vitro and the effects of uc MSC-exos on the proliferation of HSF were detected by CCK-8 assay.The effect of uc MSC-exos（2μg / ml）on HSF migration was detected by scratch test.Real-time quantitative PCR was used to detect the m RNA expression of HSFcollagen-I,fibronectin and vascular endothelial growth factor after uc MSC-exs intervention.Secondly,24 male BALB/c mice aged 8 weeks were selected to construct a mouse model of full-thickness cutaneous wounds and divided into uc MSC-exos group and PBS group by random number list.On the 0th,4th,7th,10 th and 14 th day after operation,the wound area were observed and the percentage of residual area were calculated in two groups.Tissue samples were taken at 7th and 14 th day after operation to observe the changes of skin tissue structure.Skin tissues were collected from two groups of mice and the expression levels of type I collagen、fibronectin and vascular endothelial growth factor were detected using q RT-PCR and Western blot.The expression level of CD31 was detected by immunohistochemistry.Results Under transmission electron microscope,uc MSC-exos was elliptical with a diameter of about 100 nm.Western blot showed positive expression of uc MSC-exos surface proteins CD63 and TSG101.Particle size analysis shows that 96.2 % of uc MSC-exos have a diameter distribution of 30 ～ 150 nm.One-way analysis of variance was used for comparisons among groups,LSD-t test was used to compare the two groups,,and the percentage of residual area of trauma in mice was measured by two-way ANOVA with multiple comparisons test.Non-paired t test was used for other statistical analysis.CCK-8 method showed that uc MSC-exos promoted the proliferation of HSF.（P<0.05）.The scratch test showed that compared with the PBS group,the migration ability of uc MSC-exos cells was significantly enhanced（t=19.80;P < 0.01）.The m RNA and protein expressions of type I collagen,fibronectin and vascular endothelial growth factor in uc MSC-exos group were significantly higher than those in PBS group（all P < 0.01）.The percentage of residual area in uc MSC-exos group reached（64.2±10.66）%,（43.31±7.06）%,（19.49 %±5.76）% and（0.09±0.02）% on the4 th,7th,10 th and 14 th day after operation,which was significantly lower than that in PBS group（t=2.259、3.931、3.531、4.737,P<0.05）.Uc MSC-exos promoted the expression of Collagen-I,FN,VEGF m RNA and protein in mouse skin（P <0.05）.Immunohistochemistry showed that the number of CD31 positive cells in the uc MSC-exos group was significantly higher than that in the PBS group（P < 0.05）Conclusions Subcutaneous injection of uc MSC-exos can promote acute skin wound healing in mice,which may be related to uc MSC-exos promoting the synthesis of extracellular matrix,VEGF and CD31 in wound tissue of mice,and promoting the proliferation,migration and synthesis of extracellular matrix of HSF.