摘要:
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200 μg·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200 μg·mL-1)下24 h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。200 μg·mL-1的MWCNTs处理3 h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。
Abstract:
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.