Effects Of Different Concentrations Of Hydrogen Peroxide On Adipose Stem Cells And The Mechanism Of SIRT1 In Oxidative Stress

[OBJECTIVE] Mesenchymal stem cells play an important role in tissue repair and regeneration,Stem cell senescence and exhaustion are considered important drivers of organismal aging,and stem cell supplementation can alleviate the local aging and dysfunction of organs or tissues.Therefore,stem cells have emerged as a promising cell source for cell-based therapy,and among all kinds of stem cells,adipose-derived mesenchymal stem cells have more advantages.However,senescence and low survival rate compromise the outcome of cell-based therapy resulted from oxidative stress.Adipose-derived stem cells are sensitive to reactive oxygen species(ROS).Although endogenous ROS is a critical signaling molecule,high levels of ROS may lead to cell dysfunction and cell death.But SIRT1 plays an important role in oxidative stress.In this study,we studied the characteristics of adipose derived mesenchymal stem cells,then observed the effect of hydrogen peroxide at different concentrations on adipose derived mesenchymal cells,and the mode and mechanism of SIRT1 in oxidative stress.[METHODS] 1.Adipose-derived mesenchymal stem cells were extracted from human adipose tissue.The morphology and proliferation characteristics of mesenchymal stem cells were observed.The surface markers of mesenchymal stem cells were identified by flow cytometry and the multidirectional differentiation ability of adipose-derived mesenchymal cells was identified by osteogenesis,cartilage and adipogenic differentiation experiments.2.Different concentrations of hydrogen peroxide in the adipose-derived mesenchymal cells,to observe the effects of different concentrations of hydrogen peroxide on the adipose-derived mesenchymal cells.The effects of different concentrations of hydrogen peroxide on the apoptosis of adipose-derived mesenchymal cells were detected by CCK-8,flow apoptosis cytometry and TUNEL experiments,and the effects of different concentrations of hydrogen peroxide on the senescence of adipose-derived mesenchymal cells were observed by?-galactosidase(SA-?gal)staining experiment,and autophagy related proteins and p53 associated apoptosis pathway protein and RNA expression levels were confirmed by Western blot and PCR experiments.3.To observe the effect of SIRT1 on autophagy,senescence and apoptosis of adipose-derived mesenchymal stem cells.Adipose-derived mesenchymal stem cells were exposed to SIRT1 inhibitors(EX527)or activators(SRT1720),and observed the effects of inhibition or activation of SIRT1 on autophagy and senescence of adipose-derived mesenchymal stem cells,inhibit or activate blot through western SIRT1 experimental detection,Changes of autophagy related proteins,p53 associated apoptosis pathways.Finally,CO-IP experiments were used to confirm the interaction between SIRT1 and p53 protein.[RESULTS] 1.The isolation,identification and culture of adipose-derived mesenchymal stem cells.The proportion of adipose-derived mesenchymal stem cells are relatively pure and contains almost no endothelial cells,fibroblasts and monocytes.2.Adipose-derived mesenchymal cells were exposure to different concentrations of hydrogen peroxide,and it can be found that different concentrations of hydrogen peroxide have different effects on adipose-derived mesenchymal stem cells.CCK-8,flow apoptotic cell experiment and TUNEL experiment have shown that lower concentrations of hydrogen peroxide can promote the proliferation of adipose-derived mesenchymal stem cells,and higher concentrations of hydrogen peroxide can inhibit the proliferation of mesenchymal stem cells and even promote cell death.And ?-galactosidase(SA-?gal)staining experiments have observed that lower concentrations of hydrogen peroxide can reverse the aging of mesenchymal stem cells.Western blot results show that hydrogen peroxide can promote autophagy levels of adipose-derived mesenchymal stem cells,so we suspect that hydrogen peroxide may reverse aging by increasing the autophagy levels of stem cells,and that protein levels such as p53,P21 and c-caspase3 rise as the concentration of hydrogen peroxide increases.3,And the Western blot results show that with the increase of hydrogen peroxide concentration,the protein level of SIRT1 also increased,the application of EX527 and SRT1720 confirmed that SIRT1 can affect the aging of cells by regulating autophagy pathway.And SIRT1 also plays a regulatory role in the level of p53 protein,PCR experiments show that SIRT1 has no effect on p53 mRNA level,so it is confirmed that SIRT1 may regulate the p53 level through post-transcription modification.Finally,CO-IP experiment confirmed the interaction between SIRT1 and p53 protein,and SIRT1 through deacetylation to make p53 deacetylation,deacetylation sites easy to combine ubiquitin,which can lead to p53 degradation.[CONCLUSION] 1.Mesenchymal cells isolated from adipose tissue have the characteristics of stem cells,for example multidirectional differentiation potential and the stem cells extracted from adipose tissue maintain high purity and high survival rate.2,Different concentrations of hydrogen peroxide have different effects on cells,low concentration of hydrogen peroxide can promote the proliferation of adipose-derived mesenchymal stem cells,and can increase autophagy levels by activating SIRT1 to reverse the aging of adipose-derived mesenchymal stem cells,high concentration of hydrogen peroxide to promote apoptosis.3.Hydrogen peroxide can also increase the level of SIRT1 protein and autophagy in a dose-dependent manner.4,SIRT1 can promote p53 degradation through deacetylation.