【作者】 王双英；

【导师】 朱心强；

【作者基本信息】 浙江大学 ， 卫生毒理学， 2007， 硕士

【摘要】 【目的】随着纳米技术的迅速发展及纳米材料的大量增多，纳米技术的安全性问题正引起世界范围的重点关注。多壁纳米碳圈（MWCNO）是富勒烯（C60）的同素异形体，又称为嵌套的富勒烯（nested fullerenes）。本实验使用的MWCNO是由电弧法制备的直径在30nm左右纳米材料。有研究表明碳纳米颗粒进入肺部后能转移到心血管系统，同时有人试图将其用作抗肿瘤等药物的载体，进入血液后可直接接触并作用于心血管系统，因此其安全性研究显得特别重要。Frank Chen等已就MWCNO对人皮肤成纤维细胞的细胞毒性和基因组学水平的影响进行研究，结果表明MWCNO可引起一系列与细胞代谢、凋亡、细胞周期、应激反应、细胞信号转导和炎症反应相关基因的mRNA水平的改变。但至今未见MWCNO对心血管系统影响的研究报道。磷酸化的组蛋白H2AX（γH2AX）是目前国内外研究细胞DNA损伤的热点之一，γH2AX在DNA损伤的修复以及DNA损伤位点检查点蛋白的聚集中发挥着重要的作用，特别是DNA双链的损伤。许多与染色体结构的维持、保护、修复相关的蛋白质有组织按顺序地在DNA损伤位点结合形成焦点（foci）复合物，共同完成对DNA损伤的检测和修复，并且在此过程中根据损伤程度的不同导致细胞周期停顿或细胞凋亡。因此，根据细胞在受到DNA损伤尤其是DNA双链断裂损伤后，在DSBs位点会出现由γH2AX形成的焦点这一特性，γH2AX已成为检测细胞DNA损伤的一个新的特异性指标。体内外研究发现多种纳米颗粒可以引起机体产生ROS。目前许多空气颗粒物(主要为PM_2.5、PM₁₀)和超微颗粒（UFPs）造成DNA损伤的研究都集中在氧化应激诱导的机制，认为DNA是ROS的主要靶点，ROS的产生是空气中颗粒物质引起DNA损伤的重要机制。本研究观察MWCNO是否引起血管内皮细胞凋亡、细胞周期改变、DNA损伤及其损伤机制，以评价MWCNO潜在的心血管毒性。【方法】采用体外实验的方法。用台盼蓝染色法观察不同浓度（0.2、1、5、25、50、100、200μg／ml）的MWCNO对人血管内皮细胞（HUVEC）的抑制率，根据确定的半数抑制浓度(IC₅₀)设定本实验的剂量。用检测细胞周期、凋亡的试剂盒检测不同浓度（0.2、1、5μg／ml）的MWCNO染尘后血管内皮细胞的细胞周期和凋亡的变化情况。用免疫荧光法观察不同剂量（同上）的MWCNO染尘后血管内皮细胞中磷酸化组蛋白H2AX（γH2AX）焦点形成情况，比较细胞核内焦点形成的数量及荧光强度。用DCFH-DA法检测不同剂量（同上）的MWCNO染尘后血管内皮细胞内ROS水平的变化情况。免疫荧光显微镜图片用Image Pro Plus软件进行γH2AX焦点定量计数。所有结果采用SPSS统计软件分析。【结果】流式细胞仪凋亡检测显示：1、5μg／ml的MWCNO显著促进了HUVECs细胞的凋亡，凋亡率与对照组比较有显著性差异。细胞周期检测显示，各浓度MWCNO作用后均未引起明显的细胞周期改变，G1、G2、S期细胞比例与对照组比较均无显著性差异。γH2AX识别抗体免疫荧光技术检测了MWCNO对HUVECs细胞DNA双链损伤的情况，结果发现MWCNO能诱导γH2AX焦点形成，并具有剂量和时间依赖性，高浓度（5μg／ml）MWCNO诱导的γH2AX焦点数目最多，荧光强度也最强，随着时间延长γH2AX焦点的数量及强度也随之增多、增强。各浓度MWCNO处理的HUVECs细胞12h后可引起细胞内ROS水平升高，各处理组与对照组的差异有统计学意义，处理24h后ROS有所下降，但与对照组比较仍有上升趋势，其中5μg／ml与对照组比较p=0.052。【结论】MWCNO（1-5μg／ml）能引起HUVEC细胞DNA断裂和细胞凋亡，并伴有ROS水平升高，提示其损伤机制可能与MWCNO诱导ROS增加，引起氧化损伤有关。更多还原

【Abstract】 [Objective] With the rapid development of nanotechnology, the problem of safety of nanotechnology has been concerned greatly. Carbon nanomaterials, including carbon nanoparticles and nanotubes, have been one of the most extensively used nanoparticles, because of their unique and superior properties. Multiwall carbon nano-onions （MWCNOs）, which will be the focus of this study, represent a relatively recently discovered allotrope of carbon derived from the more intensively studied fullerene (C₆₀). Giant, nested fiillerenes, generally called nano-onions, are usually produced by an underwater carbon-arc discharge. Our arc-produced MWCNOs are typically about 30nm in diameter. Recent investigations provide evidence that nanoparticles can be translocated directly from the lungs into blood circulation and extrapulmonary organs. Someone speculates that MWCNO will be effective cancer killing agents and they can directly affect blood circulation. Thus it is necessary to evaluate the biological safety of MWCNO. Frank chen et found that MWCNOs generated mRNA level changes in exposed skin fibroblasts, including changes in mRNA levels from genes involved in metabolism, apoptosis, cell cycle, stress response, cellular transport, and inflammatory response. So far, there are no relative research reports of MWCNO on the cardiovascular system.Recently the phosphorylation of histone H2AX denoted γH2AX has gained attention for its relationship with DNA damage. The phosphorylated form of histone variant H2AX plays an important role in the recruitment of DNA repair andcheckpoint proteins to sites of DNA damage, particularly at double strand breaks （DSBs）. Many relation proteins of maintaining, protecting, repairing of chromosome structure can form a foci organically, when they combine with the site of DNA damage, to check and repair the DNA damage. In this process, it can induce cell cycle to stop and cell to apoptosis according to the degree of DNA damage.Excessive generation of ROS that overwhelms the antioxidant defense system can oxidize cellular biomolecules. Free radicals generate a large number of oxidative modifications in DNA, including SB and base oxidations. The oxidative stress mediated by PM may arise from direct generation of reactive oxygen species from particles. Oxidative stress has been implicated in many diseases, including cardiovascular disease, macular degeneration, pancreatitis, and cancer. Oxidative stress-induced DNA damage appears to an important mechanism of action of urban particulate air pollution. DNA is considered to be an important target for reactive oxygen species （ROS） generated as a consequence to air pollution exposure.We investigated the influence of MWCNO on the cell cycle, apoptosis, DNA damage and its mechanism of HUVECs.[Methods] In vitro experiment, the HUVEC cells were cultured in complete 1640 medium. Trypan Blue Exclusion Test of Cell Viability was used to study the cytotoxicity of different concentration（0.2、1、 5、 25、 50、 100、 200μg/ml） of Multiwall Carbon Nano-Onions on HUVEC cells. Apoptosis and cell cycle of HUVEC were detected by flow cytometry. And immunofluorescencer was used to observe γH2AX foci formation in HUVEC cells after treated with different concentration （0.2 、 1、 5μg/ml） at different time point （6、 12、 24h）. The fluorescent intensity of reactive oxygen species （ROS） was determined by Microplate Spectrophotometer.[Results] The results showed that MWCNO failed to induce the change of cell cycle on HUVEC. The percentage of apoptosis increased with the increase of dose after the administration of MWCNO. The result from immunofluorescence microscope shown that MWCNO induced γH2AX foci formation exhibited a time-and dose-dependent manner, as the highest concentration （5μg/ml） and 24h induced the most foci. ROS levels were significantly higher than that of the controlgroup after the administration of MWCNO for 12h.[Conclussion] MWCNO induced apoptosis in HUVEC. A dose-dependent increasein the percentage of apoptosis cells was observed. Exposure to Multiwall carbonnano-onions induces DNA damage in HUVEC cells. In the meantime, the level ofROS in HUVEC cell increased after treated with MWCNO.更多还原