Therapeutic Effect And Mechanism Of Adipose-derived Stem Cells On Seawater Immersion Wounds

Background and objective:Skin wound healing is a complex process involving a series of biological processes,including repair cell proliferation and migration,wound defect re-epithelialization,collagen regeneration and neovascularization.Diabetes,obesity,persistent infection and the use of corticosteroids and other factors can make skin wounds difficult to heal,resulting in the formation of chronic skin wounds.Chronic wound refers to the wound that does not achieve anatomical and functional integrity within 30 days after injury,and eventually the patient may have serious consequences such as amputation or even death due to wound infection.At present,with the rapid development of China's maritime industry,the open injuries of maritime fighters and coastal residents are extremely vulnerable to seawater immersion.The main chemical composition of seawater is sodium chloride,and it also contains different proportions of potassium chloride,calcium chloride,magnesium chloride,magnesium sulfate and other inorganic salts.The global average salinity of seawater is 34.7,pH is 8-8.4,showing an obvious state of hypertonic alkalinity.In addition,seawater contains a large number of microorganisms,especially Gram-negative bacteria.The above characteristics determine that long-term immersion of skin wounds in seawater will lead to tissue necrosis and infection,prolong the healing time of skin wounds and make them refractory war wound.At the same time,President Xi proposed that China should build a world-class navy in an all-round way,and the scale and frequency of our army going to sea will continue to increase in the future.therefore,the treatment of seawater immersion injuries has important practical value and far-reaching historical significance for the navy.However,it is a pity that there are still few reports on the effect of seawater on skin wounds and its mechanism.Stem cell therapy has become a new direction in the treatment of chronic wounds.Adipose-derived stem cells(ADSCs)are stem cells with multi-directional differentiation potential extracted from adipose tissue.Adipose-derived stem cells can migrate to damaged parts of the skin and differentiate into accessory tissues to repair damaged skin through their multi-directional differentiation potential.At the same time,the adipose-derived stem cells have a paracrine function to secrete a variety of growth factors,thereby inhibiting the local inflammatory response of the wound,accelerating the formation of new blood vessels,and ultimately promoting wound healing.However,there are no reports on the application of adipose stem cells in seawater immersion wound repair.In this research,human umbilical vein endothelial cells(HUVEC)or human immortalized keratinocytes(HaCaT)were co-cultivated with human adipose-derived stem cells(hADSCs)in a simulated seawater environment to investigate the function of human adipose-derived stem cells on seawater-immersed cells.Subsequently,we established a seawater immersion model of mouse full-thickness skin defect,and investigated the repairing effect and its potential mechanism of seawater-immersed wounds by multi-point injection of human adipose stem cells.Methods:1.The tissues of 5 healthy women after abdominal subcutaneous liposuction were selected and then added to digestive enzyme solution,followed by digestion,centrifugation,re-suspension and other steps of plank culture.Then,the morphology of hADSCs cells was observed under microscope,the general situation of hADSCs marker(CD29,CD105,CD90,CD49d,CD45,CD34,HLA-DR)was determined by flow cytometry,and the differentiation ability of hADSCs was identified by adipogenic and osteogenic induction medium.2.The effects of different concentrations of seawater on the proliferation of HUVEC and HaCaT cells were detected by CCK-8 kit,so as to determine an appropriate concentration of seawater inhibition for follow-up research.Then,hADSCs was co-cultured with HUVEC or HaCaT cells in Transwell chamber to simulate the re-epithelialization effect and neovascularization effect of wound repair.EdU cell proliferation test and cell scratch test were used to explore the effects of seawater and hADSCs co-culture on the proliferation and migration of HUVEC and HaCaT cells.3.The model of full-thickness skin defect wound was established and divided into control group,SW group,SW+DMEM group and SW+hADSCs group.Different intervention measures were taken to observe the wound healing efficiency of the four groups.Then,the wound sections were stained with HE,Masson and immunohistochemical staining to evaluate the effect of wound repair.Finally,bioinformatics analysis was used to explore the potential molecular mechanism and Western blotting and qPCR techniques were used to detect the expression of EGFR and VEGFR proteins and the activation of MEK/ERK signal pathway in the wound.Results:1.The morphology of hADSCs cells under inverted microscope showed that hADSCs showed a long fusiform shape similar to that of fibroblasts,and the cell colonies grew radially and spirally in the process of culture.The results of multi-directional differentiation experiment showed that the extracted cells differentiated into osteoblasts and adipocytes successfully,which proved that the cells had differentiation dryness.The results of flow cytometry showed that the surface markers of CD29,CD49d,CD90 and CD 105 were strongly positive,and the expression rates were 99.4%,98.4%,99.2%and 92.9%,respectively.The surface markers of CD34,CD45 and HLA-DR were negative.2.CCK-8 experiment confirmed that the proliferation activity of HUVEC and HaCaT cells was significantly inhibited under the culture medium of 10%seawater concentration,and the inhibitory effect persisted with the extension of culture time.The results of EdU cell proliferation assay and cell scratch assay showed that the proliferation and migration of HUVEC and HaCaT cells were significantly inhibited at 10%seawater concentration.hADSCs co-culture reversed this inhibitory effect and enhanced the proliferation and migration ability of HUVEC and HaCaT cells.3.The results of in vitro experiment showed that seawater immersion significantly delayed the wound healing of full-thickness skin defect,and hADSCs injection promoted the healing efficiency of seawater immersion wound.The results of HE and Masson staining sections showed that compared with the control group and SW+hADSCs group,the effect of repairing skin thickness,regeneration of skin appendages such as hair follicles and sweat glands and collagen deposition were significantly decreased in SW group.The results of immunohistochemistry and immunofluorescence staining showed that hADSCsk could differentiate into skin stem cells and vascular endothelial cells and promote wound healing.At the same time,compared with the control group and SW+hADSCs group,the expression of Ki67,CK19 and CD31 in SW group decreased significantly,the number of proliferating cells decreased significantly,and the re-epithelialization and regenerative vascularization of wound were significantly delayed.The differential genes between hADSCs and endothelial cells were analyzed by bioinformatics,and there were 9676 differential genes.The results of GO annotation and KEGG enrichment analysis showed that up-regulated differential genes were mainly enriched in biological processes such as genetic information replication pathway,protein binding and cell mitotic cycle,while down-regulated differential genes were mainly enriched in extracellular matrix and PI3K-Akt molecular pathway.The interaction network between differential genes was analyzed by PPI network,and 20 core genes with the highest node degree were screened.The results showed that CDK1,CCNB1 and CCNA2 were the core genes with the highest degree of up-regulation of the first three nodes of differential genes,while FN1,COL1A1 and COL1A2 were the core genes with the highest degree of down-regulation of the first three nodes of differential genes.Finally,the expression of EGFR and VEGFR protein in repaired wound was detected by Western blotting.The results showed that the expression of EGFR and VEGFR protein in SW group was significantly lower than that in control group and SW+hADSCs group.The mRNA levels of type I collagen,type ? collagen and ERK pathway were detected by qPCR.The results showed that the mRNA expression of type ? collagen and type ? collagen in SW group was significantly lower than that in control group and SW+hADSCs group,and the activation of MEK/ERK signal pathway was blocked.Conclusion:High quality hADSCs were successfully obtained from adipose tissue by enzyme digestion method,which provided seed cells for follow-up experimental research.In vitro experiments showed that seawater immersion significantly decreased the proliferation and migration of HUVEC and HaCaT cells,while hADSCs co-culture could reverse this inhibition and promote the proliferation and migration of HUVEC and HaCaT cells.In vivo experiments show that seawater immersion can significantly hinder wound repair,while hADSCs can differentiate into vascular endothelial cells,skin stem cells and other cell types for repair,and can accelerate the proliferation of repair cells,wound re-epithelialization and neovascularization,and then promote the repair of defective skin tissue.In addition,hADSCs can promote the expression of EGFR and VEGFR proteins in seawater immersed wounds,thus regulating the activation of MEK/ERK pathway and promoting wound healing.