Effect Of Celastrol On Histone Release In Neutrophils And A Method Preliminarily Developed For Detection Of Phagocytosis

Objective:Observe the effect of celastrol on the expression of mTOR mRNA and the release of histone H3 in neutrophils,and explore the relationship between celastrol and histone H3,and to provide basic experimental results and theoretical basis for the clinical application of celastrol.By means of transmission electron microscopy and confocal fluorescence microscopy confirmed that graphene quantum dots can be effective uptake of neutrophils.Based on flow cytometry,a method for the evaluation of phagocytosis of neutrophil was established.The method was applied to evaluate the phagocytosis of neutrophils in healthy people and patients after chemotherapy.A new method for detecting neutrophil phagocytosis was established.Method:1.Effects of celastrol on mTOR expression and Histone H3 release from neutrophils:Different concentrations of celastrol intervened in neutrophils 1h,1.5h,2h,then the expression of mTOR mRNA was detected by Real-time PCR and then incubated with histone H3 primary antibody,secondary antibody under laser confocal fluorescence microscopy.2.Establishment the mean of graphene quantum dots evaluate neutrophil phagocytosis and preliminary application observation neutrophils phagocytosis the graphene quantum dots by transmission electron microscopy and laser confocal fluorescence microscopy.Based on the flow cytometry to explore the optimum conditions of neutrophil phagocytosis of the graphene quantum dots,then establish the method of detecting neutrophil phagocytosis and apply with the phagocytosis of neutrophils in healthy adults and patients after chemotherapy.Result:1.High concentration of celastrol can decrease the expression of mTOR mRNA.Low concentration of celastrol can promote the expression of mTOR mRNA.2.High concentration of celastrol can induce the release of histone H3,while low concentration of triptolide can inhibit the release of Histone H3.3.Compared with the healthy group,the average fluorescence intensity of the chemotherapy group was significantly lower.4.GQDs were engulfed into the cells by phagocytic vesicles and pseudopodia of neutrophils under transmission electron microscopy.5.GQDs were phagocytosed into the cells by neutrophils under confocal laser scanning microscopy.Conclusion:1.The graphene quantum dots can be used to detect the phagocytosis of neutrophils.2.The expression of mTOR mRNA and the release of Histone H3 were biotactic in different concentrations of triptolide.