The Role And Mechanism Of Exosomes Derived From Human Umbilical Cord Mesenchymal Stem Cells In The Repair Of Diabetic Ulcers

Objective: Diabetic foot ulcers（DFU）are one of the most common complications of diabetes mellitus.Existing clinical therapies cannot effectively cure DFU.In this study,exosomes derived from human umbilical cord mesenchymal stem cells（HucMSC-Ex）were used to intervene DFU model,and the function and molecular mechanism of HucMSC-Ex would be explored in order to provide a new strategy for clinical treatment of DFU.Methods: High fat diets combined with a small dose of streptozotocin（STZ）were used to establish a type 2 diabetes mellitus（T2DM）rat model,and then a full skin excision was performed on the back of each diabetic rat to establish a rapid DFU model.HucMSC-Ex and exosomes derived from human fetal lung fibroblasts-1（HFL1-Ex）were extracted and identified based on the technology established earlier in our research group.Rats with DFU were randomly divided into PBS group,HucMSC-Ex group,exosome-free conditioned medium（Ex-free CM）group,HFL1-Ex group,clinical alginate dressing group.After the operation,the wound healing process of different groups was observed at different time points.The wound closure rates were compared and analyzed.The proliferation-and-apoptosis related proteins were detected by Western blot.Skin structures were observed by HE staining.The proliferation indicator PCNA and the neovascularization indicator CD31 were detected by immunohistochemical staining.The collagen deposition was detected by Sirius red staining.According to the above experimental results,the therapeutic effects of HucMSC-Ex in the DFU model were comprehensively evaluated.Rat dermal fibroblasts（DFL）and human umbilical vein endothelial cells（HUVEC）were used as target cells in vitro.First,the target cells were stimulated with different concentrations of glucose to establish a cell model with high glucose damage,and then HucMSC-Ex was added for treatment.The effects of high glucose and HucMSC-Ex on cell viability,proliferation,migration,and tube formation ability were detected through CCK8 assay,cell colony formation assay,Transwell assay,and tube formation assay.According to the microRNAs（miRNAs）sequencing results,the miRNAs in HucMSC-Ex that may play a role in promoting DFU repair were selected.The sequencing results were verified by quantitative real-time PCR（qRT-PCR）.The downstream target gene of the selected miRNA was verified by dual-luciferase reporter gene assay in HEK293 T cells.Transfection of synthetic nucleic acid fragments in DFLs and HUVECs was used to detect the role and the molecular mechanism of the selected miRNA.The electroporation technology was used to acquire engineered HucMSC-Ex.The engineered HucMSC-Ex were applied in vivo and in vitro to explore whether it could become an optimized treatment strategy for DFU.Results: The repairing effects of HucMSC-Ex on the DFU model was significantly better than other groups,and HucMSC-Ex could protect skin cells from damages of high glucose in vitro.According to the miRNAs sequencing results,a brand new miRNA molecule—NC₀00019.10₁3474（abbreviated as miR-13474）was found enriched in HucMSC-Ex.Experiments in vivo and in vitro confirmed that miR-13474 played a key role in promoting DFU repair,and the function of miR-13474 is completed through the miR-13474 / CPEB2 / TWIST1 axis.The electroporation technology was used to import miR-13474 mimics into HucMSC-Ex,making HucMSC-Ex over express miR-13474.Experiments in vivo and in vitro showed that engineered HucMSC-Ex with over-expression of miR-13474 could be a better treatment for DFU.Conclusion: HucMSC-Ex promoted DFU repair by mediating the miR-13474 / CPEB2 / TWIST1 axis.At the same time,engineered HucMSC-Ex with overexpression of miR-13474 was expected to become an optimized treatment strategy for DFU.