Study On The Cycloheximide-induced HL-60 Cell Apoptosis And Changes Of Mitochondria

Aim:To establish the CHX-induced HL-60 cell apoptosis model and study on the change of mitochondria; to inhibit the ERK and p38 pathway respectively during the CHX-induced HL-60 cell apoptosis process in order to study on the influence of HL-60 cell apoptosis and change of mitochondria and the relationship between the cycloheximide-induced HL-60 cell apoptosis process and the the ERK and p38 pathway.Methods:To inhibit the ERK and p38 pathway respectively by PD and SB. Detecting the apoptotic and necrotic cell ratios by AnnexinV-FITC/PI double staining flow cytometry and the sub-diploid cell ratios by PI staining flow cytometry. To observe and take JC-1 staining picture and Horchest staining picture by confocal microscopyand fluorescene microscopy. To measure the J-aggregate (FL2) and J-monomer(FL1 )by JC-1 flow cytometry and calculate the △Ψ_mby(FL1/FL2 ), to analyze the relationship between the mitochondria and the physical parameter of the cell during the CHX-induced HL-60 cell apoptosis process by dividing different region of JC-1 staining cell according to the different fluorescene intensity, to divide and compare the low J-aggreagte cell of different group according to the FL-2. To measure the mitochondria mass by NAO flow cytometry and ROS producing ratios by H₂DCFDA/PI double staining flow cytometry. To measure the apoptotic cell ratios and △Ψ_m by DiOC₆(3)/Annexin V-APC double staining flow cytometry.Results:CHX can induce the HL-60 cell apoptosis. The apoptotic and necrotic cell ratios is lower than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; inhibiting p38 pathway during this process the apoptotic cell ratio has no significant difference and the necrotic cell ratios is higher compared with CHX group's. The sub-diploid cell ratio is lower than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process from 9h; The sub-diploid cell ratio is higher than the CHX group when inhibiting p38 pathway during this process from 9h. CHX can induce the Avm decrease. The △Ψ_mis higher than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; The △Ψ_m is lower than the CHX group when inhibiting p38 pathway during this process. The low mitochondria mass cell ratio of CHX group at 18h is higher than control group's, The low mitochondria mass cell ratio is lower than the CHX group's when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; the The ROS production cell ratio of CHX group at 18h is higher than control group's.Conclusions:CHX can induce HL-60 cell apoptosis, the cell mitochondria depolarization, decrease of A W m and mitochondrial mass; Inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process can lessen the decrease of apoptotic and necrotic cell ratios, the cell mitochondria depolarization and the reduction of mitochondrial mass, indicating this process is closely related to the ERK pathway. Inhibiting the p38 pathway during the CHX-induced HL-60 cell apoptosis process can increase the necrotic cell ratio, and intensify the cell A Wm decrease, indicating that the p38 pathway is closely related to the necrosis of HL-60 cell.