单壁碳纳米管诱导人支气管上皮细胞的氧化损伤和凋亡

Single-Walled Carbon Nanotubes Induce Oxidative Stress and Apoptosis of Human Bronchial Epithelial Cell Line

[目的]研究单壁碳纳米管(single-walled carbon nanotubes,SWCNTs)对人源支气管上皮细胞株(BEAS-2B)氧化应激和细胞凋亡的影响。[方法]采用生长状态良好的BEAS-2B,分别以含0、10、50、100、200μg/mL SWCNTs的培养液处理24、48h。采用四甲基偶氮唑盐(MTT)比色法检测SWCNTs对BEAS-2B细胞活力的影响;采用荧光探针2’,7’-二氯荧光素二乙酸(DCFH-DA)进行活性氧(ROS)检测;采用黄嘌呤氧化酶法测定超氧化物歧化酶(SOD)活性;采用可见光法测定过氧化氢酶(CAT)活性;采用硫代巴比妥酸(TBA)法测定丙二醛(MDA)的含量;采用二硫代二硝基苯甲酸(DTNB)法测定谷胱甘肽过氧化物酶(GSH-Px)活性;采用阳离子荧光染料罗丹明123测定线粒体膜电位;采用pNA法检测细胞内caspase-3的蛋白活性;Real-time PCR测定细胞内bax和bcl-2基因的表达变化。[结果]当处理浓度大于10μg/mL时,SWCNTs可诱导BEAS-2B细胞存活率下降,细胞内ROS含量增加,线粒体膜电位减低,MDA升高,抗氧化酶系(SOD、CAT、GSH-Px)酶活性降低,caspase-3蛋白活性增加,bax在转录水平上表达量增加,bcl-2表达量降低,各实验结果均呈效应随浓度变化的趋势(P<0.05)。[结论]SWCNTs能够诱导BEAS-2B细胞的氧化损伤和细胞凋亡。

[ Objective ] To investigate the effect of single-walled carbon nanotubes （SWCNTs） on oxidative stress and apoptosis of human bronchial epithelial cell line （BEAS-2B）. [ Methods ] BEAS-2B cells were cultured with 0, 10, 50, 100, and 200 μg/mL SWCNTs separately for 24 h and 48 h. The viability of BEAS-2B cells was detected by tetrazolium （MTT） assay; the generation of reactive oxygen species （ROS） was detected by fluorescent probe （DCFH-DA）, the enzymatic activity of super oxide dismutase （SOD） was detected by xanthine oxidase method; the enzymatic activity of catalase （CAT） was detected by visible spectrophotometry; the content of malondialdehyde （MDA） was determined by thiobarbituric acid （TBA）; the enzymatic activity of glutathione peroxidase （GSH-Px） was determined by dithio nitrobenzoic acid （I）TNB）; the mitochondrial membrane potential was determined by cationic fluorescent dye rhodamine 123; the intracellular caspase-3 protein activity was determined by pNA assay; the mRNA levels of bax and bcl-2 were determined using reverse transtription polymerase chain reaction （RT-PCR）. [ Results ] When the concentration of SWCNTs was more than 10 μg/mL, the viability of BEAS-2B cells declined, the levels of intracellular ROS increased, the mitochondrial membrane potential declined, the lipid peroxidation product （MDA） increased, the activities of antioxidant enzymes （SOD, CAT, GSH-Px） reduced, the protein activity of caspase-3 increased, the expression of bax mRNA increased while the bcl-2 mRNA reduced. All the results were exhibited in a dose-response manner （P 〈 0.05）. [ Conclusion ] SWCNTs could induce oxidative stress and apoptosis in BEAS-2B cells.