| 题名 | 氧化石墨烯等人工纳米材料对底栖动物（ Tubifex tubifex） 和哺乳动物细胞系（ Hepa细胞和 PC12细胞）的毒性研究 |
| 作者 | 张盼红 |
| 学位类别 | 硕士 |
| 答辩日期 | 2016-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 赵斌 |
| 关键词 | 人工纳米材料，底栖动物，细胞毒性，芳香烃受体 |
| 其他题名 | The toxicity study of graphene oxide and other manufactured nanomaterials on sediment invertebrate(Tubifex tubifex) and mammalian cell lines (Hepa cell and PC12 cell ) |
| 学位专业 | 环境科学 |
| 中文摘要 | 纳米材料由于其独特的特性，被广泛应用在生物医学，化妆品，再生能源，电子设备和环境修复等各种领域。在众多的纳米材料中，纳米金（ Au-NP），氧化石墨烯纳米材料（GO）和功能化多壁碳纳米管（f-MWCNT）尤其在生物医学领域应用广泛。这些纳米材料的广泛应用使得它们可能通过工业废水排放等途径释放到环境中。水体是研究纳米材料环境过程的重要介质。一旦纳米材料进入到水体，它们会经过一系列的转化，比如溶解、聚集、成团等过程，最终沉降在底泥中，因此底泥可能会成为纳米颗粒的最终聚集场所，并可对底栖动物产生不同程度的毒性效应。另外，通过食物链的传递和放大作用，纳米材料最终会进入到动物及人体并对生态系统和人类健康带来潜在的危害。但是目前应用低等和高等的动物和细胞模型综合评价纳米材料生态毒性的研究还比较欠缺。本研究拟应用底栖模式生物及哺乳动物细胞综合评价在生物医药领域应用较为广泛的纳米材料的毒理效应。研究选择了底栖模式生物 Tubifex tubifex进行生态毒理学研究，同时选取了不同组织来源的小鼠肝癌细胞（Hepa）和具有神经细胞特征的大鼠嗜铬细胞瘤细胞（PC12）评价纳米材料的细胞毒性，并应用实验室自行研发的芳香烃受体（AhR）报告基因生物检测系统初步探索了纳米材料对生物感受器 AhR的作用。纳米材料选择了 Au-NP，GO和 f-MWCNT. 本研究具体内容及结果主要包含以下两个方面： 1）研究纳米金和氧化石墨烯纳米材料对底栖动物的生态毒理效应。选用颤蚓为研究对象，对其栖息的底泥环境进行 Au-NP（4.9 ± 0.14 nm）和 GO-NPs（116± 0.05 nm）的暴露，以其规避行为，筑穴行为，生物累积量及生存率为毒性评价指标。结果表明，纳米金对颤蚓的暴露剂量在 10和60 µg/g•底泥干重范围内对其归避行为和生存率的影响是轻微的，纳米金也可以在颤蚓体内累积，并且随着暴露浓度的升高而增加。对于氧化石墨烯纳米材料，在暴露浓度为 20和 180 µg/g•底泥干重范围内对颤蚓的生存率没有影响，对其规避行为可产生轻微的影响，然而可以显著地影响其筑穴行为。而纳米金对其筑穴行为影响的环境相关性需要进一步探讨。 2）研究了两种常用的碳纳米材料对哺乳动物细胞系的毒性效应，包括GO-NPs和 f-MWCNT。首先以 ATP产生为指标考察了纳米材料对Hepa和PC12细胞的潜在细胞毒性的剂量及时间-效应关系，随后选择典型剂量考察对细胞形态的影响，同时分析上述效应的组织差异性以及与纳米材料表面修饰的构效关系。最后通过研究纳米材料对 AhR信号通路的影响，初步探讨其细胞效应的氧化石墨烯等人工纳米材料对底栖动物（Tubifex tubifex）和哺乳动物细胞系（ Hepa细胞和PC12细胞）的毒性研究潜在机制。研究对象为羧基化碳纳米管（MWCNT-COOH），氨基化碳纳米管（MWCNT-NH2），无修饰的氧化石墨烯（GO）以及聚乙二醇修饰的氧化石墨烯（GO-PEG）。纳米材料的细胞给药浓度范围为 10-150μg/mL。结果表明，上述纳米材料对细胞ATP生成的抑制作用呈现出剂量和时间效应关系，而细胞对 GO-PEG较不敏感。与 GO-PEG相比，GO在较低浓度和较短给药时间条件下，就能显著抑制细胞的 ATP产生。在 PC12细胞中 ATP发生改变所需的时间更短；相同条件下，GO可导致PC12细胞形态发生更显著的变化，说明 PC12较Hepa对GO更敏感。而MWCNT-COOH和 MWCNT-NH2纳米材料对两种细胞的 ATP生成的影响没有明显的差别。但是与ATP结果不同，仅 GO-PEG可以激活 Hepa细胞中的 AhR，说明与 AhR的作用可能是在细胞活力没有受到影响的情况下，更为敏感的生物效应指标。 综合上述结果，本研究发现不同种类的碳纳米材料对细胞的潜在毒性效应呈现出不同的在剂量/时间-效应关系及组织来源差异性。而对 AhR信号通路的影响是在细胞活力没有受到影响的情况下，更为敏感的生物效应指标。未修饰的氧化石墨烯在个体水平和细胞水平上均表现出一定的毒性。 |
| 英文摘要 | Engineered nanoparticles (ENPs) are widely applied in diverse fields, such as medicine, cosmetics, renewable energy, food industry, electronic devices and environmental remediation. Among various engineered nanomaterials, gold (Au),graphene oxide (GO) nanoparticles and functioned multi-wall nanotubes are widely used, especially in medicine. The widespread use of these nanomaterials is likely to increase their release into the environment via wastewater discharges. Once these nanoparticles are released into the aquatic environment, they will likely undergo transformation processes including dissolution, aggregation, agglomeration, and eventually settle into the sediment. Therefore, sediment may become an ultimate reservoir for ENPs. As a result, nanoparticles may be ingested by deposit- feeding benthic invertebrates and potentially be bio-magnified within the food chain, which may pose a high risk to invertebrates and higher trophic level organisms. However,few studies have been conducted on the ecotoxicity of nanomaterials on organisms. In the present study, sediment invertebrate Tubifex tubifex is employed to study the eco-toxicity of Au-NPs and GO-NPs. At the same time, Hepa cell and PC12 cell are employed to assess the cytotoxicity of carbon nanomaterials. Furthermore, the effect of nanomaterials on the AhR signaling pathway is also discussed using the AhR report gene biological detection system. Au-NP, GO and f-MWCNT are employed in the present study. The study includes the following parts: 1) Evaluate the ecotoxicity of sediment associated gold - and graphene oxide nanoparticles on sediment dowelling invertebrates Tubifex tubifex. In this part,mortality, behavioral impact (only GO-NP) and uptake (only Au-NP) of sediment-associated Au-NPs (4.9 ± 0.14 nm) and GO-NPs (116 ± 0.05 nm) to T.tubifex were assessed in a number of 5 day exposure experiments. The results showed that the applied Au-NP exposure (concentration ranging from 10 to 60 µg Au/g dry weight sediment) had no adverse effect on T. tubifex survival, while Au bioaccumulation increased with exposure concentration. In the case of GO-NPs,no mortality of T. tubifex was observed at a concentration ranging from 20 to 180 µg GO /g dw sediment, whereas burrowing activity was significantly reduced. Our results suggest that the short-term exposure of Au-NPs at 60 µg Au /g has no obvious toxic effect on T. tubifex while GO-NPs at 20 - 180 µg GO /g, can cause toxicity to T. tubifex by affecting its behavior. Our results also show that burrowing behavior is a more sensitive endpoint when evaluating the ecotoxicity of nanomaterial on invertebrates. Further studies should focus on the effect of Au-NP on the burrowing behavior of T. tubifex. 2) In this part, we focus on the cytotoxicity of carbon nanomaterials (GO-NPs and f-MWCNT) to two different kinds of mammal cell lines (Hepa cell and PC12 cell).First, we chose the ATP production as an indicator to examine the cytotoxicity of nanomaterials as a function of dose and time. Then, we used the typical treatment dose to observe the morphology of cells, as well as to investigate the discrepancy among different functioned carbon nanomaterials. Finally, we investigated the effect of nanomaterials on the AhR signaling pathway in order to explore the mechanism of toxicity induced by carbon nanomaterials. Four different carbon nanomaterials including carboxylated carbon nanotubes (MWCNT-COOH),amino carbon nanotubes (MWCNT-NH2), non-modified graphene oxide (GO) and polyethylene glycol-modified graphene oxide (GO-PEG) with concentrations of 10 - 150 µg/mL were employed. The results showed that the effect of carbon nanomaterials on the cellular ATP production exhibit a dose and time dependent manner, whereas the nanomaterials are less sensitive to GO-PEG. Compared with GO-PEG, GO can significantly inhibit ATP production at lower concentrations and shorter time, wherein the less time was needed in PC12 cell compared with Hepa cell. Under the same conditions, the morphology of PC12 cell changed a lot after GO treatment, indicating that PC12 is more sensitive to GO than Hepa cell.In addition, there was no significant difference on ATP production between MWCNT-COOH and MWCNT-NH2, whereas only GO-PEG could activate the AhR signal pathway in Hepa cell, suggesting that AhR is a more sensitive indicator when the cell viability is not affected. Based on these results, we conclude that the toxicity of carbon nanomaterials is dependent on the dose, treatment time and tissues. However, AhR is a more sensitive indicator when the cell viability is not affected by nanomaterials. The toxicity of GO exist both in individual and cell levels. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/37052]  |
| 专题 | 生态环境研究中心_环境化学与生态毒理学国家重点实验室 |
| 推荐引用方式 GB/T 7714 | 张盼红. 氧化石墨烯等人工纳米材料对底栖动物（ Tubifex tubifex） 和哺乳动物细胞系（ Hepa细胞和 PC12细胞）的毒性研究[D]. 北京. 中国科学院研究生院. 2016. |
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