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目的研究 p38对放线菌酮(CHX)经线粒体途径诱导 HL-60细胞死亡的影响。方法利用 p38持异性阻断剂 SB203580(SB)阻断 HL-60细胞 p38途径,分别设对照组、单纯 SB 组、CHX组和 CHX 联合 SB 组;于处理6,9,12,18,24 h 利用碘化丙锭(PI)染色流式细胞术检测亚二倍体峰细胞比率;于处理6和18 h 进行膜联蛋白Ⅴ(Annexin Ⅴ)/PI 双标流式细胞术测定凋亡和坏死细胞比率;于处理18 h 利用 JC-1染色流式细胞术分析,通过对 FL2的划分比较不同处理时间高 JC-1集合体量细胞比率;测定细胞 JC-1集合体(FL2)和 JC-1单体(FL1)平均荧光强度,并计算线粒体膜电势(△Ψ_m,FL2/FL1)。结果在 CHX 处理6 h HL-60细胞亚二倍体峰细胞比率显著高于对照组,在此过程中阻断 p38途径则在处理9 h 开始亚二倍体峰细胞比率显著高于 CHX 组。处理18 h 凋亡细胞比率:CHX组[(27.9±0.52)%]和 CHX 联合 SB 组[(28.54±1.38)%]均显著高于对照组[(2.45±0.65)%](P<0.01);CHX 联合 SB 组与 CHX 组比较差异无统计学意义(P>0.05)。处理18 h 坏死细胞比率:CHX 组和 CHX 联合 SB 组均显著高于对照组(P<0.01);CHX 联合 SB 组显著高于 CHX 组(P<0.01)。此外,CHX 组和 CHX 联合 SB 组高 JC-1集合体细胞比率均显著低于对照组(P<0.01),CHX联合 SB 组高 JC-1集合体细胞比率显著低于 CHX 组(P<0.01)。处理18 h 时各实验组△Ψ_m CHX 组(0.17+0.01)和 CHX 联合 SB 组(0.05±0.003)均显著低于对照组(0.38±0.02)(P<0.01),CHX 联合 SB 组△Ψ_m 显著低于 CHX 组(P<0.01)。结论 CHX 可诱导 HL-60细胞凋亡和线粒体去极化,在该过程中阻断 p38途径可使线粒体的去极化作用增强,本模型中 p38途径可能与 HL-60细胞的坏死密切相关。
Objective To investigate the effect of p38 on the death of HL-60 cells induced by mitochondrial pathway of cycloheximide (CHX). Methods The p38 pathway of HL-60 cells was blocked by SB203580 (SB), an isoprotectant of p38. The control group, SB group, CHX group and CHX combined with SB group were respectively set as control group. After 6, 9, 12, 18 and 24 h The ratio of sub-diploid peak cells was detected by flow cytometry with propidium iodide (PI) staining. Apoptosis and necrosis were detected by annexin Ⅴ / PI double-flow cytometry at 6 and 18 h after treatment The percentage of cells in JC-1 aggregates was determined by JC-1 stained flow cytometry at 18 h after treatment. FL-2 cells were treated with different concentrations of JC-1 aggregates for different treatment times. 1 monomer (FL1) average fluorescence intensity, and calculate the mitochondrial membrane potential (△ Ψ_m, FL2 / FL1). Results The ratio of sub-diploid peak cells in HL-60 cells treated with CHX for 6 h was significantly higher than that in control cells. In the process of blocking p38 pathway, the ratio of sub-diploid cells was significantly higher than that of CHX cells after 9 h of treatment. Compared with control group [(2.45 ± 0.65)%] (P <0.01), the ratio of apoptotic cells in 18 h treatment group was significantly higher than that in control group [(27.9 ± 0.52)%] and CHX combined with SB group (28.54 ± 1.38% There was no significant difference between CHX combined SB group and CHX group (P> 0.05). The proportion of necrotic cells treated for 18 h was significantly higher in CHX group and CHX combined with SB group than that in control group (P <0.01). CHX combined SB group was significantly higher than CHX group (P <0.01). In addition, the ratios of high JC-1 aggregates in CHX group and CHX combined with SB group were significantly lower than those in control group (P <0.01), and the proportion of high JC-1 aggregates in CHX group and SB group was significantly lower than that in CHX group (P <0.01) ). At 18 h, the △ Ψ_m CHX group (0.17 + 0.01) and the CHX combined SB group (0.05 ± 0.003) were significantly lower than the control group (0.38 ± 0.02) (P <0.01) Lower than CHX group (P <0.01). Conclusion CHX induces apoptosis and mitochondrial depolarization in HL-60 cells. Blocking the p38 pathway may enhance mitochondrial depolarization in this process. The p38 pathway in this model may be closely related to the necrosis of HL-60 cells.