Changes in cell cycle,apoptosis,phagocytic function,and expression of inflammatory factors in human monocyte/macrophage cell line Rho0 constructed from THP-1 cells with mtDNA depletion
Objective To observe the changes in the cell cycle,apoptosis,phagocytic function,and expression of inflammatory factors in human monocyte/macrophage cell line Rho0 constructed from human monocytic leukemia cells(THP-1 cells)with mitochondrial DNA(mtDNA)depletion.Methods We took THP-1 cells,and used ethidium bro-mide(EB)to remove mtDNA in the cells to construct the new human monocyte/macrophage Rho0.Rho0 cells were treat-ed with LPS(1 mg/mL)or solvent stimulation for 24 h,respectively,which were taken as the Rho0-LPS group and Rho0-solvent group.Untreated THP-1 cells(Control group)were stimulated with LPS(1 mg/mL)or solvent for 24 h respective-ly,which were taken as the Control-LPS group and Control-solvent group.Flow cytometry was used to detect the proportion of cell cycle in each group after PI staining.The number of apoptotic cells was measured by Annexin V-FITC/PI method.The phagocytosis function of cells in each group was evaluated by Red-Zymosan dye phagocytic test(mean fluorescence in-tensity).The expression of inflammatory cytokines(IL-1α,IL-1β,TNF-α,IL-6,IL-8,IL-10,IL-12 mRNA)was detect-ed in Rho0 cells(Rho0 group)and control cells(Control group)by RT-qPCR.Results Compared with Control-solvent group,the proportions of cells in the G0-G1 phase,S phase and G2-M phase in the Rho0-solvent group decreased,the num-ber of apoptotic cells increased and the mean fluorescence intensity of phagocytic dyes increased;the number of apoptotic cells and the mean fluorescence intensity increased in the Control-LPS group(all P<0.05).Compared with Rho0-solvent group,the proportion of cells in the G0-G1 phase decreased and the number of apoptosis increased in the Rho0-LPS group(all P<0.05).Furthermore,compared with the control group,the mRNA expression levels of IL-1β,TNF-α,IL-8 and IL-10 increased in the Rho0 group(all P<0.05).Conclusion Removal of mtDNA of mononuclear/macrophage can cause the cell cycle arrest of Rho0 cells,promote the apoptosis,and enhance phagocytic function and proinflammatory function.
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