活性氧参与多壁碳纳米管诱导的RAW264.7细胞毒性

Involvement of Reactive Oxygen Species in Multi-Walled Carbon Nanotubes(MWCNTs)-Induced Cytotoxicity to RAW264.7 Cells

碳纳米管以其独特的结构和性能,在生物医药和电子等领域广泛应用,而其生态安全性也成为科学界关注的焦点。为探究多壁碳纳米管(MWCNTs)诱导的细胞毒性机制,将小鼠肺泡巨噬细胞(RAW264.7)暴露于6个浓度梯度(0、25、50、100、150和200μg.mL-1)的MWCNTs中,应用噻唑蓝(MTT)法测定细胞存活率,用2’,7’-二氯荧光素二乙酸(DCFH-DA)荧光染色法测定细胞内活性氧的生产量,用流式细胞方法测定MWCNTs对细胞周期的影响。同时使用抗氧化剂氮乙酰半胱氨酸(NAC)验证MWCNTs诱导的细胞氧化损伤的作用机理。结果显示,MWCNTs对RAW264.7的细胞毒性呈剂量依赖性。暴露于不同浓度的MWCNTs(25、50、100、150和200μg.mL-1)下24h后,细胞活力分别为对照的74%、62%、59%、51%和45%。MWCNTs对RAW264.7的周期阻滞作用主要发生在G0/G1期。200μg.mL-1的MWCNTs处理3h后活性氧较对照组上升6.6倍。NAC对MWCNTs细胞毒作用有明显的抑制作用,且NAC能减弱MWCNTs对RAW264.7的细胞周期阻滞作用。研究表明,活性氧能够介导MWCNTs对小鼠巨噬细胞RAW264.7的损伤,并且MWCNTS通过细胞周期G0/G1期的阻滞,诱导细胞凋亡。

Carbon nanotubes with their unique structure and function are widely applied in biomedical and electronic industry.Simultaneously,the issue concerning ecological safety of carbon nanotubes is becoming a hot topic in the academic field.To study the mechanism of cytotoxicity induced by multi-walled carbon nanotubes（MWCNTs）,alveolar macrophages of mouse（RAW264.7 cells） were exposed to MWCNTs with six concentrations（0,25,50,100,150 and 200 μg·mL-1）.The cell viability was detected by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method.The content of reactive oxygen species（ROS） in cells was measured by flow cytometer using florescent dye 2＇,7＇-dichlorodi-hydrofluorescein diacetate.Cell cycle arrest and induced apoptosis were analyzed by flow cytometer.Antioxidant N-acetylcysteine（NAC） was used to verify the mechanism of oxidative damage of cell induced by MWCNTs.Results showed that the cytotoxicity of MWCNTs to RAW264.7 cells was in a dose-dependent manner.After exposure to MWCNTs（25,50,100,150 and 200 μg·mL-1） for 24 h,cell viability decreased to 74%,62%,59%,51% and 45% of the control,respectively.MWCNTs could induce cell cycle arrest mainly in the G0/G1 phase.ROS content in cells increased 6.6 times of the control after RAW264.7 cells exposure to 200 μg·mL-1 MWCNTs for 3 h.NAC could significantly inhibit the cytotoxicity induced by MWCNTs,and decrease the retardation on the cell cycle of RAW264.7 cells.Therefore,it is demonstrated that MWCNTs induced damage to RAW264.7 cells through ROS-mediated mechanism,then resulted in G0/G1-phase cell cycle arrest and cell apoptosis.