摘要
为了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,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;用透射电子显微镜和光学显微镜观察细胞结构的变化;用DNA凝胶电泳、细胞DNA含量、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。结果表明:SNP能抑制HL-60细胞生长,典型的细胞形态改变、DNA片段化、亚二倍体峰比例增加、DNA末端标记和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)活性降低。ROS含量和CAT、GST、GPX活性与SNP之间也有明显的量效关系。结论:细胞内活性氧自由基增加和抗氧化能力下降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。
This study was aimed to investigate the changes of reactive oxygen species and antioxidative capacity on nitric oxide induced apoptosis in HL-60 cells. By means of in vitro incubation of HL-60 cells with sodium nitroprusside（SNP）, the growth inhibition was detected by MTT assay. Cell morphology was observed by transmission electronmicroscopy and light microscopy. The apoptosis was analyzed by DNA agarose gel electrophoresis, DNA content and Annexin-V/PI labeling method. Reactive oxygen species （ROS） labeled with dihydrorhodamin 123 in cells was determinated 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）. The results indicated that SNP could inhibit HL-60 cell growth. Cell apoposis was confirmed by typical cell morphology, DNA fragment, sub-G1 phase and Annexin-V/PI labeling method. HL-60 cell apoptosis was induced by SNP in a dosage- and time- dependent manner. After exposing to SNP at the concentration of 0.5 - 3.0 mmol/L for 48 hours, the mean fluorescence intensity of ROS in cells was significantly higher than those in groups control and potassium ferricyanide （PFC）. During the apoptosis process, level of ROS in cells increased, levels of GSH, CAT,GPTand GPX decreased. The significant doseeffect relationship existed between the levels of ROS, CAT, GST, GPX and SNP dose. It is concluded that change of intracellular reactive oxygen species and antioxidative capacity are an important factors during the process of SNP-induced apoptosis in HL-60 cell.
出处
《中国实验血液学杂志》
CAS
CSCD
2005年第4期579-583,共5页
Journal of Experimental Hematology
基金
浙江省医学科学研究基金资助项目
编号2002A0010
关键词
一氧化氮
硝普钠
细胞凋亡
HL-60细胞
活性氧
抗氧化能力
nitric oxide
sodium nitroprusside
apoptosis
HL-60 cell
reactive oxygen species
antioxidative capacity
