Establishment and Phenotypic Identification of MSCs Senescence Model Induced by Hydrogen Peroxide
Objective To establish a stable cell aging model induced by hydrogen peroxide(MSCs),and to verify the effect of hydrogen peroxide on MSCs aging,so as to establish a stable MSCs aging model.Method To induce the third generation of human umbilical cord mesenchymal stem cells by hydrogen peroxide for 48 hours.The morphological changes of the cells were observed by light microscope,the cell proliferation curve was drawn by CCK-8 experiment,the content of reactive oxygen species(ROS)in ROS experimental cells,β-galactosidase staining and alkaline phosphatase(Alkaline phosphatase were detected.ALP)staining and activity assay were used to determine the changes of osteogenic differentiation,and Western blotting electrophoresis(Western blot,WB)was used to detect the changes of aging-related proteins.Results Under the intervention of hydrogen peroxide(H2O2),the morphology of cells in the experimental group changed and flattened,the proliferation rate of MSCs cells decreased significantly,the content of ROS increased significantly,the positive cells of β-galactosidase staining increased,the positive region of ALP staining decreased,the activity of ALP decreased,and the expression of senescence-related proteins such as P16,P21 and p53 increased.Conclusion The aging of MSCs induced by hydrogen peroxide was verified.It was confirmed that a concentration of 200 µm hydrogen peroxide could construct a stable in vitro senescence model,which provided a reference for exploring the senescence mechanism of MSCs.
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