不同荧光探针检测微囊藻毒素-LR诱导的氧自由基

Microcystin-LR induces the generation of oxygen free radical determined by different fluorescent probe

目的研究微囊藻毒素-LR(MC-LR)对人肝细胞内超氧阴离子自由基(O2.-)、羟自由基(.OH)水平的影响,探讨不同荧光探针的检测效果。方法调整人肝癌HepG2细胞密度为5×104/mL,分别设终浓度为10,30,100μmol/L的MC-LR染毒组和去铁敏(DFO,1 mmol/L)对照组以及MC-LR(100μmol/L)+DFO(1 mmol/L)染毒组,另设溶剂对照组(0.1%DMSO),培养4 h后应用荧光探针氢化乙啶(HE)和2-[6-(4'-羟基)苯氧基-3H-占吨醇-3-on-9-yl]-苯甲酸(HPF)分别检测细胞内O2.-和.OH含量。结果 30～100μmol/L的MC-LR染毒4 h后细胞内O2.-和.OH含量明显高于溶剂对照组(P<0.05),染毒剂量与.OH含量之间呈现剂量-反应关系,去铁敏和MC-LR联合染毒组的O2.-和.OH含量明显低于100μmol/L MC-LR染毒组(P<0.05)。结论 MC-LR可引起人肝癌HepG2细胞内O2.-和.OH含量升高,用荧光探针HE和HPF能够快速、灵敏地检测细胞内的超氧阴离子自由基和羟自由基水平。

Objective To study the generation of superoxide and hydroxyl radical in human liver cells treated with Microcystin-LR（MCLR） determined by different fluorescent probe.Methods Human hepatoma cell lines HepG2 were treated with MCLR,then the generation of superoxide and hydroxyl radical was determined by the hydroethidine（HE） and 2-[6-（4＇-hydroxy） phenoxy-3H-3-on-9-yl]benzoic acid（HPF）.Results After exposure to 30 μmol/L and higher concentrations of MC-LR for 4 h,the generation of superoxide and hydroxyl radical in MC-LR-treated group was significantly higher than that of solvent control group（P0.05）.The generation of superoxide and hydroxyl radical in the group that received combined treatment with deferoxamine（DFO） and MC-LR was significantly lower than that of 100 μ mol/L MC-LR-treated group（P0.05）.Conclusion MCLR induces the generation of superoxide and hydroxyl radical in human hepatoma cell lines.Fluorescence probe HE and HPF is reliable and sensitive method for detecting the superoxide and hydroxyl radical.