摘要：
目的 研究1,4苯醌(1,4-BQ)能否诱导HL60细胞发生自噬以及活性氧(ROS)在自噬发生中的作用.方法 CCK8法检测不同浓度1,4-BQ对HL60细胞的毒性作用,并据此选择观察细胞自噬效应的适宜染毒浓度.将对数生长期细胞分为对照组、1,4-BQ染毒组(10 μmol/L 1,4-BQ染毒24 h)、N-乙酰半胱氨酸(NAC)组(5 mmol/L NAC处理24 h)和1,4-BQ+NAC干预组(5 mmol/L NAC干预1h后加10 μmol/L 1,4-BQ处理24 h).吖啶橙染色检测自噬酸性囊泡,免疫荧光染色法定性检测自噬蛋白LC3的表达定位,蛋白免疫印迹法定量检测LC3和Beclin1的蛋白表达.结果 HL60细胞活力随着1,4-BQ浓度增加而逐渐下降,呈剂量-反应关系,与对照组比较,20.0、40.0 μmol/L染毒组HL60细胞存活率明显降低,差异有统计学意义(P＜0.05).与对照组比较,1,4-BQ染毒组自噬酸性囊泡增多;免疫荧光定性检测结果显示,膜型LC3II蛋白表达增加.蛋白免疫印迹结果显示,1,4-BQ染毒组LC3和Beclin1蛋白表达增加,分别比对照组增加了12.4％和27％,差异有统计学意义(P＜0.05).与1,4-BQ染毒组比较,1,4BQ+NAC干预组自噬酸性囊泡明显减少,LC3和Beclin1蛋白表达下降,分别比1,4-BQ组下降了12.6％和22.6％,差异有统计学意义(P＜0.05).结论 1,4-BQ可诱导HL60细胞发生自噬,其诱导的自噬至少部分来源于ROS途径,抗氧化剂可有效抑制自噬的发生.%Objective To investigate whether autophagy can be induced by 1,4-benzoquinone (1,4BQ) in HL60 cells,as well as the role of reactive oxygen species (ROS)in induced autophagy.Methods In order to determine a suitable 1,4-BQ treatment concentration for autophagy detection in HL60 cells,the cell vitality were examined by CCK8 assay.Logarithmic-growth-phased cells were divided into control group,1,4-BQgroup(10μmol/L 1,4-BQ,24 h),NAC group (antioxidant n-acetyl cysteine,5mmol/L,24 h) and the 1,4-BQ+NAC group (5 mmol/L NAC were preincubated for 1h prior to the treatment with 10 μmol/L 1,4-BQ for 24 h).The autophagic acidic vesicle were inspected by acridine orange staining,LC3 were detected by immunofluorescence staining,and expressions of LC3 and Beclin1 were quantitatively detected by Western blot.Results The results from cell viability test indicated that 1,4-BQ exhibited a dose-dependent toxicity to HL60cells.Compared with control group.the cell viability in 20.0、40.0μmol/L concentration were decreased obviously,and the differences had statistical significance (P＜0.05).Compare with contrd group acidic vesicle,LC3II,LC3II/LC3I and Beclin1 protein expressions were increased in 1,4-BQ group,after both respectively 12.4％ and 27％,the differences had statistital significance.While 1,4-BQ+NAC group was observed that acidic vesicle,LC3 and Beclin1 protein level were markedly lower than 1,4-BQ group,after both decreased 12.6％ and 22.6％ respectively,both the difference were statistically significant (P＜0.05).Conclusion 1,4-BQ can induce autophagy in HL60 cells,the induction of autophagy is at least partly resulted from ROS.Antioxidant can effectively suppress the occurrence of induced autophagy.
