The Study Of Hydrogen Peroxide-induced Apoptotic Stem Cells Regulating Macrophage Polarization

ObjectiveStem cells have good self-renewal and multidirectional differentiation potential.Some cells may undergo apoptosis after stem cell replacement,and there are few studies on the pathway of stem cell transformation after apoptosis.Macrophages are the main phagocytes responsible for the removal of dead cells in most organs.Under the action of different cytokines,macrophages can be polarized into M1/M2 type.There are few studies on the polarization of macrophages by the apoptotic stem cells mentioned above.Therefore,in this study,the optimal concentration of hydrogen peroxide-induced apoptosis of mesenchymal stem cells was selected in vitro to explore the changes in the secretion function of cells induced by apoptosis and the effect on the polarization of macrophages.Methods1.BMSCs were successfully isolated and cultured.BMSCs were identified as having high purity and good differentiation potential,which could be used for later experimental study.2.Hydrogen peroxide with a concentration of 0.15%-0.6% was used to induce apoptosis of BMSCs,and the optimal concentration for inducing apoptosis of BMSCs was preliminarily screened by q PCR semi-quantitative detection.3.Flow cytometry,Hoechst 33258 staining and TUNEL cell apoptosis staining were used to detect the apoptosis of BMSCs.4.The secretion of the corresponding cytokines: TGF-?1,IFN-?,TNF-? and IL-10 in hydrogen peroxide-induced BMSCs was detected by q PCR technique.5.The apoptotic BMSCs were co-cultured with macrophages,and the polarization of macrophages to M1/M2 type was detected by q PCR technique.Results1.In this experiment,we have successfully isolated and cultured rat bone marrow mesenchymal stem cells,and BMSCs are identified to have a good differentiation level,which can be used for later experimental studies.2.The appropriate concentration of hydrogen peroxide(0.15%-0.6%)can induce apoptosis of BMSCs,and the optimal concentration is 0.3%.3.The apoptosis model of BMSCs induced by 0.3% hydrogen peroxide was successfully established in this experiment,and the apoptosis of BMSCs showed a significant time dependence.4.In the process of detecting the secretion level of BMSCs after apoptosis,it was observed that the secretion level of TGF-?1 and TNF-? increased with time,and IL-10 did not show a time dependence.5.The results of macrophage polarization showed that the expression of M2-type related cytokines in macrophages was significantly increased and the expression of M1-type related cytokines was decreased after co-culture of apoptotically induced BMSCs and macrophages,suggesting that macrophages could polarize to M2-type macrophages.ConclusionIn this study,it was confirmed that the appropriate concentration of hydrogen peroxide(0.15%-0.6%)could induce apoptosis of BMSCs in vitro,and the optimal concentration of apoptosis induction was 0.3%.After apoptosis induction,BMSCs were activated and their paracrine levels changed significantly.Subsequent experiments also proved that cytokines secreted after the apoptosis of BMSCs can induce the occurrence of M2-type polarization of macrophages.