| Objective:(1)Co-culture human epidermal keratinocyte(HEK)and human umbilical mesenchymal stem cell conditioned medium(Human Umbilical-derived Mesenchymal Stem Cell,HUMSC-CM)to explore whether it can develop epithelial-mesenchymal stem cells Epithelial Mesenchymal Transformation(EMT).(2)Explore the possible molecular mechanism of HUMSC-CM in the process of epithelial-mesenchymal transformation.(3)To verify the molecular mechanism of HUMSC-CM to accelerate wound healing in vivo.Materials and methods:(1)After separating,culturing and purifying human umbilical cord mesenchymal stem cells(Human Umbilical-derived Mesenchymal Stem Cells),use adipogenic,osteogenic,and chondrogenic differentiation media to determine their tri-lineage differentiation potential and use flow cytometry Detect specific markers(CD14,CD29,CD34,CD44,CD45,CD90)and characterize them;select human epidermal keratinocyte(HEK)cells as experimental objects,collect human umbilical cord mesenchymal stem cells and culture them After finding out the appropriate concentration of HEK,the proliferation and migration ability of HEK were tested by MTT method,scratch test,and transwell test;Western blot was used to determine the relevant indicators of EMT(Ecadherin,?-SMA,Slug)for detection,and the application of cellular immunofluorescence technology to detect changes in the expression level of E-cadherin,Slug,?-catenin,and ?-SMA in HEK,so as to explore the EMT of HUMSC-CM on HEK cells The role of change.(2)Detect the content of TGF-?1 in the conditioned medium by using the flow cytometry multiple protein quantitative technique;After the small molecule drug TGF-?1 and its inhibitor SB435142 were given to HEK,the EMT-related molecules were detected by Western blotting(E-cadherin,?-SMA,Slug)protein expression levels;at the same time,the use of lentiviral packaging technology to establish a HEK knockout Smad2 stable cell line,to further explore the role of TGF-?1/smad signaling pathway in mediating EMT-like transformation;Use cellular immunofluorescence technology to detect changes in the expression levels of E-cadherin,Slug,?-catenin,and ?-SMA in HEK,and explore the role of TGF-?1/smad signaling pathway in the process of EMTlike transformation of keratinocytes;use scratch experiment,The transwell experiment verifies the change of HEK's migration ability after EMT.(3)Use female nude mice BALB/c to establish a full-thickness skin injury model for random grouping.The wound was injected with HUMSC-CM as the treatment group,and the Epilife basal medium group as the control group.,12 days to collect images of wound healing,use Image J software to determine the wound healing rate;cut the wound tissue for H&E staining,compare the wound healing of the treatment group and the control group,and measure the indications including epithelial structure and epidermal thickness,Inflammatory response and vascular regeneration,etc.,so as to explore the role and mechanism of HUMSC-CM in wound repair.Results:(1)The three-line differentiation induction experiments of umbilical cord mesenchymal stem cells are all positive,and the percentages of negative markers detected by flow cytometry are CD14(0.42%),CD34(0.6%),and CD45(0.11%).And the positive markers CD29(93.99%),CD44(96.69%),CD90(98.44%)prove that the proposed cells are umbilical cord mesenchymal stem cells;MTT experiments show that HUMSC-CM can promote the proliferation of HEK cells and scratches Experiments and transwell experiments show that HUMSC-CM can promote the migration ability of cells.Western blot results show that after adding HUMSC-CM,the expression of HEK's epithelial marker E-cadherin is inhibited,while the interstitial markers ?-SMA and Slug.The expression increased,and the immunofluorescence results showed that after HUMSC-CM treatment,the expression of epithelial markers E-cadherin and ?-catenin was inhibited,while the interstitial markers Slug and ?-SMA were promoted,that is,HEK cells had EMT Change.(2)HUMSC-CM related molecule content determination found that the content of TGF-?1 is the highest;Western blot results show that after adding small molecule compound TGF-?1 in vitro,the process of cell EMT-like transformation is promoted,adding TGF-? inhibitor After SB435142,the process of EMT-like transformation was inhibited;after knocking out Smad-2 on HEK cells,the process of EMT-like transformation was also inhibited.After adding the small molecule compound TGF-?1,the EMT inhibition process was slightly alleviated;cell immunofluorescence results It is shown that after treatment with the small molecule compound TGF-?1,the expression of epithelial markers E-cadherin and ?-catenin are inhibited,while the interstitial markers Slug and ?-SMA are promoted,that is,HEK cells have EMT-like changes.And the process of EMT-like transformation is promoted;scratch experiments and transwell experiments show that the addition of small molecule compound TGF-?1 can significantly promote the migration ability of cells,which further proves that TGF-?1 in HUMSC-CM leads to HEK cell development.EMT-like change.(3)The wound model of mice confirmed that the wound healing speed of mice was accelerated after adding the conditioned medium of umbilical cord mesenchymal stem cells;immunohistochemical staining found that the thickness of the wound surface of the mice in the treatment group with HUMSC-CM Significantly higher than the control group,the structure of the skin is more complete;CK14 positive cells are mainly concentrated in the basal layer and sub-basal layer of the epidermis.The epidermal cell proliferates actively and the epidermal layer thickens.As time goes by,CK14 positive cells Shows a trend of migration to the wound center.On the 3rd day of treatment,a CK14-positive epidermal cell layer was seen under the blood crust in the treatment group and a clear CK14-positive cell layer under the blood crust in the control group.Conclusion: In this study,the molecular mechanism of the conditioned medium of umbilical cord mesenchymal stem cells in promoting skin wound healing in mice was preliminarily explored,and it was found that the umbilical cord mesenchymal stem cells activate the TGF-?/smad signal in the residual keratinocytes of the wound margin through paracrine action.It promotes the EMT process,accelerates the migration of wound-edge cells to the wound,accelerates re-epithelialization and wound healing,and provides a theoretical basis and basis for the wide application of umbilical cord mesenchymal stem cells in various clinical wound treatments,and provides a basis for subsequent changes.In-depth exploration of the molecular mechanism of wound healing provides new feasible ideas. |
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