一氧化氮供体诱导HL-60细胞凋亡过程中活性氧自由基和抗氧化能力的变化

The change of reactive oxygen species and antioxidative capacity on nitric oxide induced apoptosis in HL-60 cells

目的研究外源性一氧化氮(NO)供体硝普钠(SNP)诱导HL-60细胞凋亡过程中活性氧自由基和抗氧化能力的变化。方法将HL-60细胞与SNP在体外培养,用MTT法观察:NO对细胞的抑制作用;用透射电镜和光镜观察细胞结构改变,用DNA 凝胶电泳、细胞DNA含量、DNA片段原位末端标记、Annexin-V/PI法等分析细胞凋亡;用双氢罗丹明123(DHR)标记、流式细胞仪检测细胞内活性氧(ROS),并同时测定细胞内谷胱甘肽(GSH)和3种抗氧化酶的活性。结果SNP能抑制HL-60细胞生长, 典型的细胞形态改变、DNA片端化、亚二倍体峰比例增加、DNA末端标记、Annexin-V+/PI-表达增加等证实NO能诱导白血病细胞凋亡,且两者之间有明显的量效和时效关系。在此过程中,细胞内ROS水平增加,细胞内谷胱甘肽和过氧化氢酶(CAT)、谷胱甘肽S转移酶(GST)、谷胱甘肽过氧化物酶(GPX)降低。ROS和CAT、GST、GPX的含量与SNP两者之间也有明显的量效关系。结论细胞内活性氧自由基和抗氧化能力平衡的改变在外源性一氧化氮供体诱导HL-60细胞凋亡中发挥了重要作用。

Objective TO investigate the change of reactive oxygen species and antioxidative capacity on nitric oxide induced apoptosis in HL-60 cells. Methods Incubation of HL-60 cells with sodium nitroprusside (SNP)in vitro. The growth inhibion was analysed by MTT assay. Cell morphology was observed under transmission electromieromicscope and microscope.The apoptosis was analysed by DNA agarose gel electrophoresis, DNA content, TdT-mediated dUTP nick, end labeling(TUNEL)and annexin-V/PI labeling method.Reactive oxygen species (ROS) labeled with dihydrorhodamin 123 in cells was analysed by flow cytometry. The SNP-treated cells were examined for glutathione(GSH) level and activity of catalase(CAT),glutathione S-transferase(GST)and glutathione peroxidase(GPX).Results SNP can inhibit HL-60 cell growth. Cell apoposis was comfirmed by type cell morphology and DNA fragment and sub-G1 phase and TUNEL and Annexin-V/PI Labeling method.SNP induced HL-60 cell apoptosis in a dosage-and time-dependent manner.After exposing to SNP at the concentration of 0.5-3.0 mmol/L SNP for 48 h, the mean fluorescence intensity of ROS in cells was significantly higher than those in control and potassium ferricyanide (PFC). During the apoptosis process, it showed a decrease of GSH, CAT, GPT, and GPX . The level of GSH and antioxidative enzyme was decreased in a SNP dosage dependent manner. Conclusion The change of intracellular reactive oxygen species and antioxidative capacity is an important factor during the process of SNP induced apoptosis in HL-60 cell.