| Background:As the largest organ of human body,skin maintains the balance of internal and external environment.When the normal anatomical structure and functional integrity of the skin are disturbed,a series of physiological and pathological repair mechanisms related to wound healing will be initiated under the protection mechanism of the body itself.Healing is a complex and dynamic cellular signal response,which is a biological and molecular activity mainly composed of the overlapping stages of hemostasis,inflammation,proliferation,migration and remodeling to achieve tissue repair.After the injury,adipose tissue can promote the complete recovery of the original tissue structure,and its mechanism may be related to the abundant secretory bodies contained in ADSCs,which prove that cell proliferation,differentiation,migration and improvement of the relationship between cell and extracellular microenvironment play an important role;Exosomes have the same function as stem cells,which are rich in protein,mRNA,miRNA and other substances,A few miRNAs have been reported to play a role in the healing process.In recent years,miRNA has become a hot topic in research,but there are few studies on the role of miRNA secreted by exosomes in the healing process of skin damage.Objective:To investigate the role and mechanism of mir-486-5p secreted by ADSCs exosomes in the healing of skin injury.Methods:The main experiments used in this project include mouse skin injury model,tissue immunofluorescence and HE staining,Masson trichrome staining,cell transfection technology,CCK8,Western blotting,Scratch test,migration experiment and tubule formation experiment.Results:1.ADSCs derived exosomes promote skin wound healing and angiogenesisIn this part of the experiment,ADSCs were firstly isolated from the surgical tissues,and the related proteins(CD105,CD44,CD73,etc.)were detected.The result of alizarin red,oil red O,alcian blue staining showed that the ADSCs cells we isolated had the potential to differentiate into osteoblasts,adipocytes and chondrocytes.The particle size of exosomes and electron microscopy showed that particles were exosomes.In addition,HE staining results showed that the wound skin of mice injected with exosomes was basically closed,and new skin,hair follicles and fat cells had appeared,while PBS group had no significant change.The results of Masson trichrome staining showed that compared with PBS group,the collagen fibers on the surface of wound skin in exosomes group increased significantly.CD31 positive cells and vascular tissue were increased in the exocrine injection group.2.MiR-486-5p promotes the proliferation,metastasis and angiogenesis of HSF and HMEC cellsIn this study,PKH67 was used to label ADSCs deriven exosomes,and miR-486-5p mic or inhibitor was transfected by gene transfection technology.The results showed that the exosomes derived from ADSCs were engulfed by HSF and HMEC cells,and the expression of miR-486-5p in HSF and HMEC cells was increased.In addition,compared with miR-486-5p inhibitor or PBS group,we used miR-486-5p mimic or ADSCs derived exosomes and found that the ability of cell transfer was significantly enhanced.For the CCK8 experiment,it was found that ADSCs derived exosomes could promote the proliferation of HSF,and miR-486-5p inhibitor could inhibit the viability of cells.For the tubule formation experiment,it was found that the length and branches of tubules in the group of ADSCs exosomes and miR-486-5p mimic were significantly increased,while the tubules in the group of miR-486-5p inhibitor group were significantly weaker.3.MiR-486-5p secreted by ADSCs-derived exosomes promotes the proliferation,migration and angiogenesis of HSFs and HMECs by inhibiting the expression of Sp5The results of double luciferase reporter gene experiment showed that when miR-486-5p mimic and sp5 3’-UTR wild-type plasmids were co transformed,thefluorescence signal was significantly forced,but the fluorescence signal in co-transformed mimic control and sp5 3’-UTR mutation group was unchanged.In addition,when miR-486-5p mimic and inhibitor were transfected into HSF cells and HMEC cells,the expression of miR-486-5p and Sp5 protein increased,while that of miR-486-5p and Sp5 protein increased.In scratch test,compared with pcDNA-3.1control group,Sp5 overexpression plasmid significantly reduced cell mobility,while ADSCs derived exosomes significantly increased cell mobility compared with SP5 overexpression plasmid and PBS,while cell proliferation showed a similar trend;the ability of tubule formation of SP5 overexpression plasmid cells is weakened,but after the treatment of exosomes,the ability of vascular formation of cells is increased.4.MiR-486-5p promotes skin healing in miceThe skin injury model was divided into three groups: ADSCs exosomes +miR-486-5p NC group,PBS + miR-486-5p NC group and PBS + miR-486-5p antagonists group.The skin wound healing rate of exosomes + miR-486-5p NC group was the fastest,followed by PBS + miR-486-5p NC group and PBS + miR-486-5p antagonist group.HE staining showed that the number of new skin,new hair follicles and fat cells increased significantly in the exosomes + miR-486-5p NC group,but these new tissues were less in the PBS + miR-486-5p antagonists group.The results of quantitative analysis of neoepithelia and scar showed that the width of scar and neoepithelia in exosomes + miR-486-5p NC group was significantly larger than that in miR-486-5p NC group.Immunofluore-scence showed that the CD31 fluorescence in the skin of the exosomes + miR-486-5p NC group was significantly enhanced,while the CD31 fluorescence in the miR-486-5p antagonist group was weak.5.MiR-486-5p regulates the expression of CCND2 by combining Sp5,and then promotes angiogenesisAfter the treatment of ADSCs derived exosomes,the expression of compared with mir-486-5p In the group of inhibitor or CCND2 protein was significantly increased compared with PBS treatment.After the treatment of PBS and siCCND2,the expression of CCND2 protein in cells was significantly reduced,but after the treatment of exosomes,the expression of CCND2 was increased.In addition,scratch test and migration assay showed that the migration ability of cells treated bysiCCND2 and PBS decreased significantly,while the migration ability of cells treated by exosomes increased;similarly,we found that CCK8 experiment showed similar performance;For the tubule formation experiment,we found that siCCND2 treatment can inhibit the formation of tubules,the length and number of tubules.After the treatment of exosomes,the ability of tubule formation is increased.Conclusion:1.ADSCs-derived exosomes promote skin wound healing and angiogenesis2.MiR-486-5p promotes the proliferation,metastasis and angiogenesis of HSF and HMEC cells3.MiR-486-5p secreted by ADSCs-derived exosomes promotes the proliferation,migration and angiogenesis of HSFs and HMECs by inhibiting the expression of Sp54.MiR-486-5p promotes skin healing in mice5.MiR-486-5p regulates the expression of CCND2 by combining Sp5,and then promotes angiogenesis... |
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