| Background:The skin as the body’s barrier is crucial for maintaining homeostasis.Once the skin is damaged by external risk factors,the body will immediately start the process of wound healing and seal the skin defect as soon as possible to maintain the body’s homeostasis.Any factor hindering the normal progress of wound healing can lead to delayed healing or non-healing,resulting in chronic non-healing wounds,which place a heavy burden on patients.Therefore,ensuring the normal process of wound healing is very important.In general,wound healing is mediated by a variety of cells,among which Epidermal stem cells(ESCs)play an important role in this process.However,in chronic wounds,the level of oxidative stress is elevated due to iron overload,which further leads to an increased level of lipid peroxidation in ESCs,impairing the normal function of ESCs and impeding the process of wound healing.Therefore,exploring the key factors affecting the lipid peroxidation level of ESCs and elucidating the selfprotection mechanism of ESCs against iron-dependent oxidative stress can provide new ideas for the clinical promotion of wound healing.Objective:To investigate the differentially expressed proteins in ESCs under various concentrations of holo-transferrin,then search for key factors to investigate their effects on the function of ESCs,especially on the lipid peroxidation levels.Methods:1.Extraction and identification of mouse ESCsESCs were extracted from the skin of newborn mice by the type Ⅳ collagen adhesion method.The normal morphology of the extracted ESCs was observed by inverted phasecontrast microscopy and the surface markers of ESCs,including CK14,CK15,p63,CK10,were identified by Western blot analysis,flow cytometry and immunofluorescence techniques.2.Effects of changes in holo-transferrin concentration on Fe2+ and lipid peroxidation levels in ESCsESCs were cultured for two days in the medium with different concentrations(0μg/mL,1 μg/mL,10 μg/mL,100 μg/mL,1 mg/mL)of holo-transferrin,the levels of Fe2+ and lipid peroxidation in ESCs under different culture conditions were measured by flow cytometry.3.TMT quantitative proteomics analysis of differentially expressed proteinsESCs were cultured for two days in a medium without or with 100 μg/mL of holotransferrin,and TMT quantitative proteomic analysis was used to screen for differentially expressed proteins and identify key proteins.The Western blot analysis was performed to verify their expression.4.Adenoviral transfection of ESCs to achieve the overexpression of ACOT7The ESCs were transfected with empty vector adenovirus and adenovirus carrying Acot7 respectively,as the control group and the overexpression experimental group.Then the viral transfection efficiency was verified by Western blot analysis and immunofluorescence.5.Effects of overexpression of ACOT7 on the proliferation and apoptosis of ESCsCell proliferation was detected and analyzed by the IncuCyte S3 Live-Cell Analysis System to investigate the effect of ACOT7 on the proliferative capacity of ESCs.Apoptosis was detected by Annexin V-PE/7-AAD staining and flow cytometry to study the effect of ACOT7 on the apoptotic function of ESCs.6.Effects of overexpression of ACOT7 on the lipid peroxidation levels and cell viability of ESCsESCs were stimulated with H2O2,Erastin,and RSL3 for 6 h,the content of MDA and the level of lipid peroxidation in ESCs were measured by malondialdehyde(MDA)assay kit and flow cytometry to verify the effect of ACOT7 on lipid peroxidation in ESCs.The effect of H2O2,Erastin and RSL3 on the cell viability of ESCs was measured by CCK-8 assay to investigate the effect of overexpression of ACOT7 on the survival of ESCs.7.Statistical analysis All data were statistically analyzed by GraphPad Prism version 8.0.1 software,quantitativedata were expressed as mean ± standard deviation.Differences between groups were compared by the unpaired t-test,differences were considered to be statistically significant when P was less than 0.05.Results:1.Characterization of mouse ESCsThe ESCs were tightly arranged under the microscope,with an overall "pavement-like"shape and the cell shape was irregularly polygonal.The cells obtained were highly expressed in CK14,CK15,p63 and rarely in CK10,which ensured the purity of the ESCs.2.Effects of changes in holo-transferrin concentration on Fe2+ and lipid peroxidation levels in ESCsAs the concentration of holo-transferrin increasing in the medium,the level of Fe2+ in ESCs showed a tendency to increase,but there was no significant difference in the level of lipid peroxidation.Every group of ESCs was given 500μM H2O2 for 6 h respectively,and the lipid peroxidation level still remained with no significant difference.3.TMT quantitative proteomics analysis of differentially expressed proteinsThe differentially expressed protein ACOT7 was screened by proteomic analysis.Western blot analysis confirmed that the expression of ACOT7,FTH,and FTL was positively correlated with the concentration of holo-transferrin in the culture medium.4.Adenoviral transfection of ESCs to achieve the overexpression of ACOT7An overexpression adenovirus vector of ACOT7 was constructed and transfected into ESCs.Western blot analysis and immunofluorescence confirmed the transfection efficiency.5.Effects of overexpression of ACOT7 on the proliferation and apoptosis of ESCsCell proliferation and apoptosis assays confirmed that overexpression of ACOT7 had no effect on the proliferative and apoptotic functions of ESCs.6.Effects of overexpression of ACOT7 on the lipid peroxidation levels and cell viability of ESCsOverexpression of ACOT7 under the stimulation of H2O2,Erastin and RSL3 reduced the level of MDA and inhibited lipid peroxidation in ESCs,which in turn increased the survival rate of ESCs.Conclusion:As the concentration of holo-transferrin in the culture medium increased,the level of Fe2+and the expression of ACOT7 in ESCs increased.With the overexpression of ACOT7,the lipid peroxidation was inhibited,and finally the survival rate of ESCs elevated. |
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