Effects Of H₂O₂ Intervention On Reactive Oxygen Levels Of Mesenchymal Stem Cells From Different Sources

Objective:1.Human endometrial mesenchymal stem cells?hEMSCs?were isolated in vitro,and hEMSCs,human adipose-derived mesenchymal stem cells?hADSCs?,and humanumbilical cord mesenchymal stem cells?hUCMSCs?for identification of mesenchymal stem cells.2.Exploring the best concentration of DCFH-DA probe.3.To study the effect of H₂O₂ on the levels of ROS in hEMSCs,hADSCs and hUCMSCs.Methods:1.Acquisition and identification of hEMSCs,hADSCs,and hUCMSCs:hEMSCswere extracted from endometrial tissue by enzymatic digestion.Obtain recovery hADSCs and hUCMSCs from cooperative units.The three cell morphologies were observed,and three cell surface specific antigens were detected by flow cytometry.The three cells were tested for their ability to differentiate into adipocytes,osteoblasts,and chondroblasts.2.The fourth generations of hEMSCs,hADSCs,and hUCMSCs were added,and DCFH-DA probes of different concentrations were added for 30 min.The green fluorescence percentage of the cells was determined by flow cytometry to determine the optimal concentration.3.The fourth generations of hEMSCs,hADSCs,and hUCMSCs were used,and different concentrations of H₂O₂ were added as experimental groups.The DCFH-DA probes in the DCF kit were incubated for 30 minutes.After a total of 1 hour of intervention,the cell8morphology was observed using an inverted phase contrast microscope,and flow cytometry was used.The green fluorescence intensity of cells was measured,that is,the expression of ROS.4.Data analysis were performed using GraphPad Prism 8.0 Data was presented in figure and text as mean±standard deviation??±?.Differences between the two groups were compared by one-way analysis of variance,and P<0.05 indicates that the difference was statistically significant.Results:1.hEMSCs were extracted by enzymatic digestion,and hEMSCs,hADSCs,and hUCMSCs were identified.All three cells showed fibroblast-like morphology.Surface specific antigens detected by flow cytometry showed:CD73,CD90,CD105 were positive;CD34,CD45,CD11b,CD19,and HLA-DR were negative.The results of differentiation induction experiments showed that they all had the ability of adipogenic,osteogenic and chondroblastic cells.The above results indicate that the obtained cells meet the minimum standards for mesenchymal stem cells.2.Set different DCFH-DA fluorescent probe concentration gradients?0,100,150,200,250,300?M?,and explore the appropriate concentration to accurately reflect the ROS level of the cells.The experimental results show that in the three cell experiments of hEMSCs,hADSCs,and hUCMSCs,as the concentration increases,the percentage of fluorescence intensity gradually increases,reaching the plateau phase at a concentration of 250?M.3.After 1 hour of 0,50,100,200,and 300?M H₂O₂ intervention,the ROS content of both hEMSCs and hUCMSCs cells increased gradually with increasing concentrations,and increased significantly after 300?M H₂O₂ intervention;hADSCs after intervention with different concentrations of H₂O₂ There was no significant change in ROS expression.It shows that hADSCs are less affected by H₂O₂ and have a stronger tolerance,which may be related to their antioxidant capacity.Conclusion:1.HEMSCs,was isolated and cultured successfully,and hEMSCs,hADSCs and hUCMSCs were identified.The results showed that the three kinds of cells had normal morphology,conformed to the standard of MSCs surface specific antigen,and had the ability of multi-directional differentiation.2.Determine the concentration of 250?M DCFH-DA fluorescence probe as the standard concentration for follow-up experiments.3.Comparing hADSCs,hUCMSCs,and hEMSCs,hADSCs were least affected by H2O2,followed by hEMSCs,and hUCMSCs responded most strongly.Intervention at a concentration of 300?M can significantly increase the ROS levels of hUCMSCs and hEMSCs.