| Background and Objectives:Skin,the largest organ of human,is composed of the epidermis,dermis,and subcutaneous tissue,which plays a role in regulating the temperature of the human body,maintaining a stable internal environment,and preventing the entry of microorganisms and chemicals.Skin injuries including burns,trauma,diabetic foot and many other types of trauma caused by external damage or disease can be fatal.Skin burns are common and extremely complicated injuries caused by heat,light,electricity,radiation,and chemicals on the human body,such as skin or mucous membrane damage.Moreover,the subcutaneous tissue may be damaged in some severe cases.At present,deep degree skin burns are difficult to be healed with the wounds heal slowly,resulting in new skin tissue having no sweating function,lacking elasticity,and often accompanied by scar formation.Therefore,it is urgent to find a new method to treat skin burns and increase skin wound healing rate.Mesenchymal stem cells,a type of adult stem cells,are a group of stem cells derived from the mesenchymal stroma of tissues that are capable of self-renewal and multi-directional differentiation.In recent years,studies have shown that mesenchymal stem cells can produce and secrete a variety of cytokines and promote skin injury repair.Human amniotic mesenchymal stem cells(hAMSCs)are a new type of stem cells derived from newborn amniotic membranes,which have the advantages of convenient collection,strong proliferation ability,no ethical obstacles,and safety and no tumorigenicity.Whether and how hAMSCs can be used for repair of skin burns are still unknown.This study aims to explore the potential and mechanism of hAMSCs in the treatment of deep second-degree skin burns,which provides experimental evidences for finding new methods and new targets for treating skin burns.Methods:1.Isolation,cell culture and identification of hAMSCs:1)hAMSCs from amnion were isolated using trypsin and collagenase for cell culture;2)Detection of hAMSCs surface molecular markers by RT-PCR,flow cytometry and immunofluorescence;3)Osteogenic and adipogenic differentiation were analyzed to determine the multi-directional differentiation potential of hAMSCs in vitro;4)The tumorigenicity of hAMSCs in vivo and in vitro was measured using soft agar colony formation experiments and NOD-SCID mouse in vivo transplantation experiments.2.The possible mechanisms of hAMSCs and hAMSC-CM for promoting the repair of deep Ⅱ degree burn wound:1)Construct a mouse model with deep second-degree burn wound by using a homemade skin scald device;2)In situ injection of PBS,PKH26-labeled hAMSCs,H-DMEM and hAMSC-CM near the skin burn wounds,and the wound area of mice in different groups at different time points were calculated;3)The survival,migration and differentiation of hAMSCs in deep second-degree burn wound mice were detected by whole-body fluorescent imaging and immunofluorescence;4)HE staining,TUNEL apoptosis staining,PCNA and CK19 immunohistochemistry and western blot were used to analyze the apoptosis and proliferation of heat-damaged skin cells in the skin tissue of mice of different groups.3.Molecular mechanisms of hAMSCs and hAMSC-CM in inhibiting apoptosis and promoting proliferation of thermally injured skin cells:1)A thermal damage model of epidermal cells HaCAT and dermal fibroblasts DFL were constructed in vitro.A Transwell co-culture chamber was used as a co-culture system of hAMSCs,HaCAT and DFL cells;2)Annexin V/PI flow cytometry apoptosis assay,CCK8 test,scratch test were conducted to detect the apoptosis,proliferation and migration of heat-damaged skin cells in normal culture group,heat injury group,hAMSCs co-culture group and hAMSC-CM treatment group after heat injury;3)Western blot analysis was used to detect the expression of PI3K/AKT and Wnt/β-catenin signaling pathway-related proteins in heat-injured skin cells of normal culture group,heat injury group,hAMSCs co-culture group and hAMSC-CM treatment group;4)AKT inhibitor LY294002 and β-catenin inhibitor ICG001 were added to the co-culture system of hAMSCs and skin cells.Then,flow cytometry and Western blot assays were used to detect the role of hAMSCs in inhibiting the apoptosis of thermally injured skin cells and promoting their proliferation;5)The expression levels of 440 cytokines in hAMSC-CM were detected by antibody chip to screen candidates that may activate PI3K/AKT signals.Results:1.hAMSCs possessed the characteristics of the mesenchymal stem cells:1)hAMSCs isolated and cultured from amniotic membrane by two-step digestion were characterized with typical morphology of mesenchymal stem cells and strong reproduction ability;2)the results from RT-PCR,flow cytometry and immunofluorescence detection assays showed that hAMSCs expressed the mesenchymal stem cell marker genes such as CD105,CD29,CD73,CD90 and embryonic stem cell marker genes such as Oct-4,Nanog,SSEA-4,however,did not express the hematopoietic stem cell marker genes CD45,CD133,CD34 and the major histocompatibility class Ⅱ antigen HLA-DR.Major histocompatibility class I antigen HLA-ABC was found to be expressed at a lower level,but the co-stimulatory molecules CD80,CD86 and CD40 were not detected in hAMSCs;3)hAMSCs can be differentiated into osteocytes and adipocytes in vitro;4)hAMSCs had no tumorigenicity in vivo and in vitro.2.hAMSCs and hAMSC-CM transplantation in vivo promoted the repair of deep second-degree skin burns via inhibiting the apoptosis and promoting the proliferation of thermally injured skin cells:1)A mouse model with deep second-degree burn wound was successfully prepared with treatment of 80℃water for 100s;2)Calculation of wound area and wound healing rate showed that hAMSCs and hAMSC-CM transplantation in vivo promoted skin wound healing in the mouse model;3)Whole-body fluorescent imaging and immunofluorescence detection result showed that hAMSCs could survive over 21 days in mouse skin burns parts without differentiated into other cell types along with the gradually decreases of the cell numbers;4)TUNEL apoptosis detection result showed that hAMSCs and hAMSC-CM could inhibit the apoptosis of the thermally injured skin cells.Immunohistochemistry and western blot results showed that hAMSCs and hAMSC-CM could promote the proliferation of the thermally injured skin cells.3.hAMSCs and hAMSC-CM inhibited apoptosis and promoted proliferation of the thermally injured skin cells through activating PI3K/AKT and Wnt/β-catenin signaling pathways:1)HaCAT and DFL cell thermal damage models were successfully prepared in vitro by treatment of 43℃ for 50min;2)Co-culture of hAMSCs and hAMSC-CM treatment in vitro could inhibit the apoptosis of thermally injured skin cells and promote their proliferation and migration;3)hAMSCs inhibited the apoptosis of the thermally injured skin cells through activating PI3K/AKT rather than Wnt/β-catenin signaling pathway.However,hAMSCs-induced activation of PI3K/AKT signaling pathway,in turn,activated Wnt/β-catenin signaling pathway which promoted the proliferation of thermally injured skin cells;4)Cytokine antibody array showed that hAMSCs promoted the repair of skin burns through secreting a series of cytokines including PAI-1,G-CSF,Periodin,and TIMP-1 which might activate the PI3K/AKT signaling pathway.Conclusions:1.hAMSCs have the characteristics of strong proliferative ability,expressions of mesenchymal stem cell and embryonic stem cell marker genes,non-expressions of hematopoietic stem cell marker genes,low immunogenicity,multi-directional differentiation potential,and inability of tumorgenicity in vivo and in vitro.2.The in situ transplantation of hAMSCs and hAMSC-CM promotes the repair of deep second-degree skin scald through paracrine effect,in which the possible mechanism is related to their inhibition of the apoptosis and promotion of the proliferation of the heat-damaged skin cells.3.hAMSCs-derived cytokines such as PAI-1,G-CSF,Periodin and TIMP-1,may be responsible for their repair of skin injury,in which the cytokines-mediated activation of the PI3K/AKT signaling pathway inhibits the heat-injured skin cell apoptosis,while the activation of PI3K/AKT pathway further activates the Wnt/β-catenin signaling pathway,and then promotes the proliferation of thermally injured skin cells. |
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