| Objective: To investigate the effects and mechanisms of Dia-Exos on human umbilical vein endothelial cell function and wound healing in mice.Methods: The Dia-Exos was extracted from the peripheral blood of patients with diabetic foot by high-speed centrifugation.The morphology and size of Dia-Exos as well as the marker protein on the cell membrane were identified by transmission electron microscopy,dynamic light scattering,and WB.Fluorescence-labeled Dia-Exos was cocultured with endothelial cells to observe the phagocytosis of Dia-Exos by endothelial cells.The effects of Dia-Exos on endothelial cell function were observed by CCK-8,flow cytometry,migration assay,qRT-PCR,WB,and tube forming assay.The skin defect model was established in mice,and Dia-Exos was injected around the wound to observe wound healing.On the 10 th day,the blood perfusion of the wound surface was observed using animal Doppler ultrasonography.H&E staining and CD31 staining were performed on the14 th day to observe the wound tissue and neovascularization,respectively.Then,we further studied the effect of Dia-Exos-derived miR-24-3P on endothelial cells and wound healing.Blood samples of patients with diabetic foot,Dia-Exos and 14-day neonatal skin tissue of Dia-Exos group mice were taken,and the level of miR-24-3P was detected by qRT-PCR.AgomiR-24-3p was selected to simulate endogenous miR-24-3p.CCK-8,flow cytometry,migration test,qRT-PCR,WB and tube forming experiments were used to observe the effect of elevated miR-24-3p level on endothelial cell function.Then Antagonir-24-3P was used to inhibit intracellular miR-24-3p expression and to observe whether inhibition of miR-24-3p expression affected the inhibition of Dia-Exos on endothelial cell function.The skin defect model was established in mice,and AgomiR-24-3p or Antagonir-24-3P was injected around the wound to observe the wound healing.On the 10 th day,the blood perfusion of the wound surface was observed using animal Doppler ultrasonography.H&E staining and CD31 staining were performed on the 14 th day to observe the wound tissue and new blood vessels,respectively.Finally,the mechanism of Dia-Exos-derived miR-24-3P affecting wound healing was explored.The potential target genes of miR-24-3P were predicted by using the prediction website of target genes of network miR-24-3P.The binding of the target genes to MIRI-24-3P was evaluated by using a double luciferase experiment.qRT-PCR was used to detect the effect of high expression of miR-24-3P on target genes.Si RNA was used to knock down the expression of target genes in endothelial cells,and CCK-8,flow cytometry,migration experiment,qRT-PCR,WB and tube forming experiment were used to observe the effect of knocking down target genes on endothelial cell function.Results: Dia-Exos was cup-shaped or spherical in shape,and the particle size was within 30–150 nm.WB results showed the presence of the marker proteins TSG101 and CD9 on the surface of Dia-Exos.Cell experiments showed that Dia-Exos could be taken up by endothelial cells,and inhibit the proliferation,migration and tube-forming ability of endothelial cells,and promote apoptosis.Animal experiments showed that Dia-Exos could inhibit wound healing in mice,reduce local blood perfusion,and reduce neoangiogenesis.qRT-PCR results showed significantly increased levels of miR-24-3p in blood samples from patients with diabetic foot ulcer,in Dia-Exos,and in 14-day skin tissues from mice in the Dia-Exos group.AgomiR-24-3p can inhibit the proliferation,migration and tube-forming ability of endothelial cells,and promote apoptosis.AntagomiR-24-3p could reverse the inhibitory effect of Dia-Exos on endothelial cell function.In animal experiments showed that AgomiR-24-3p could inhibit wound healing of mice,reduce local blood perfusion,and reduce neoangiogenesis.Prediction site results showed that PIK3R3 may be a potential target gene for Dia-Exos-derived miR-24-3p to affect wound healing.qRT-PCR results showed that high levels of miR-24-3p inhibited the expression of PIK3R3,and in vitro experiments showed that knockdown of PIK3R3 expression inhibited the function of endothelial cells,and the addition of AntagomiR-24-3p reversed this inhibition.Conclusion: Dia-Exos can inhibit the proliferation,migration and tube-forming ability of endothelial cells,promote apoptosis,and reduce wound angiogenesis,thereby delaying wound healing.miR-24-3p in blood of patients with diabetic foot ulcer and Dia-Exos is significantly and highly expressed.Dia-Exos-derived miR-24-3p can inhibit the function of HUVECs cells,reduce local angiogenesis of the wound,and delay the healing of the wound.Dia-Exos-derived miR-24-3p delayed wound healing by targeting PIK3R3. |
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