| 题名 | 单壁碳纳米管和氧化石墨烯的免疫毒性效应与机制研究 |
| 作者 | 汪子霞 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 万斌 |
| 关键词 | 碳纳米材料 carbon nanoparticles 巨噬细胞 macrophage 细胞毒性 cytotoxicity 细胞自噬 autophagy 溶酶体 lysosome |
| 其他题名 | Immune toxicity effect and mechanism research of single-walled carbon nanotubes and graphene oxides |
| 中文摘要 | 自身不同于宏观材料的物理化学特性使得纳米材料在电子技术、环境治理以及生物医学等领域具有广泛的应用。其中，碳纳米材料由于其巨大的比表面积、很强的吸附性能及其独特的光热性质使得其在药物载体、生物成像和基因治疗等方面具有很广的应用前景。巨噬细胞作为生物体内一类重要的免疫细胞，能够识别并吞噬包括纳米材料在内的外源性颗粒物，成为纳米材料在生物体内的主要靶向细胞。本论文在细胞和细胞器水平上，研究比较了单壁碳纳米管（SWCNT）和氧化石墨烯（GO）两种碳纳米材料对巨噬细胞的免疫毒性效应及对细胞功能的影响作用，在此基础上进一步在细胞和蛋白水平上深入探讨了两种碳纳米材料对细胞自噬过程的影响。 首先，以小鼠原代巨噬细胞（PMQ）为免疫细胞实验模型，研究了SWCNT和GO的细胞毒性效应以及对细胞功能的影响。SWCNT和石墨烯经混酸处理后得到的酸化单壁碳纳米管（AF-SWCNT）和氧化石墨烯（GO），经电镜、zeta电位、红外光谱等方法表征发现，AF-SWCNT和GO的水溶液具有很好的稳定性。流式细胞仪实验结果发现，≤50μg/mL AF-SWCNT和GO不会造成PMQ细胞显著的凋亡和坏死，但是扫描电镜SEM观察发现AF-SWCNT和GO在10μg/mL 时就会显著改变细胞形态，破坏细胞膜完整性，在细胞膜周围出现大量空泡，这与LDH实验检测结果也有很好的吻合。细胞器水平的研究发现，低剂量（≤5μg/mL）的AF-SWCNT和GO便会显著降低细胞线粒体膜电位（MMP）。此外，与对照组相比较，1-50μg/mL AF-SWCNT在4小时内对胞内ROS的产生无明显影响，但50μg/mL GO在30min时便会显著增加胞内ROS的含量。对PMQ的细胞功能研究发现，AF-SWCNT和GO的暴露会导致细胞吞噬荧光纳米微球的能力（包括吞噬比例和吞噬效率）受到明显抑制，且呈明显的剂量-效应关系。在以上细胞毒性效应的实验中，GO的作用效果均强于AF-SWCNT。 在上述实验结果的基础上，仍以原代巨噬细胞PMQ为细胞模型，继续研究AF-SWCNT和GO两种纳米材料对细胞自噬的影响。细胞切片透射电镜观察发现，与对照组相比，AF-SWCNT和GO都会造成胞内自噬体的累积，增加EGFP-LC3融合蛋白的绿色荧光聚集，显著提高LC3II/LC3I的比值。但是，自噬相关的胞内基底蛋白p62的含量却显著升高。使用自噬上游通路抑制剂3MA预处理细胞后发现，与对照组相比，自噬泡的累积并没有得到明显的缓和。高分辨透射电子显微镜（HR-TEM）发现，两种纳米材料通过内吞作用进入PMQ细胞溶酶体，降低溶酶体的移动性，增加溶酶体膜的渗透性，改变溶酶体内的酸性pH降解环境。这些实验结果表明，纳米材料很可能是通过影响自噬下游的溶酶体通路造成自噬体累积的。 |
| 英文摘要 | Nanomaterials have been applied in many fields, such as electronics, environmental protection and biomedical, due to their unique properties that different from macro-materials. Among them, carbon nanomaterials have great potential in drug carrier, bioimaging and gene therapy because of huge surface area, strong adsorption ability and distinctive photothermal properties. Macrophages, the important immune cells in organic system, can defend and scavenge foreign invasion particles including nanomaterials and therefore become the main target of nanoparticles. In this paper, the toxicity and effect on cell function of AF-SWCNT and GO were investigated on both cellular and organelle levels, and the effects of both nanomaterials on autopgagy were further studied on cellular and protein level. Firstly, we studied the toxic effects of AF-SWCNT and GO on murine primarily macrophages. The AF-SWCNT and GO solutions treated with mixture acid were well-dispersed analysed by electron microscope, zeta potential and infrared spectroscopy. Flow cytometry analyses showed that with ≤50μg/mL AF-SWCNT and GO treatment , no significant cytotoxicity on macrophages was found, but SEM discovered cell morphology changed a lot, membrane integrity were lost and a large number of vacuole were emerged when exposed with 10μg/mL AF-SWCNT or GO, which coincided with LDH assay very well. ≤5μg/mL of AF-SWCNT or GO treatment damaged the mitochondrial membrane potential (MMP) on the organelle level. In addition, compared with control, 1-50μg/mL AF-SWCNT did not increase the production of ROS for 4h treatment, while 50μg/mL GO increased intracellular ROS significantly exposed at 30min. Functional analyses revealed that the phagocytosis (include phagocytic ratio and phagocytic efficiency) for latex beads of PMQ were inhibited significantly treated with AF-SWCNT and GO in does- response relation. GO was more potent than AF-SWCNT in the all mentioned tests. Based the above results, we further studied the effect of both nanomaterials on PMQ autophagy. High-resolution TEM discovered that AF-SWCNT and GO caused the accumulation of autophagosomes, increased the aggregation of EGFP-LC3 fusion protein and enhanced LC3II/LC3I ratio compared with control. However, the content of substrate protein p62 related to autophagy rose. After pretreated with the inhibitor of autophagy upstream pathway, the accumulation of autophagosomes was no obvious alleviation. TEM images also revealed both nanomaterials entered macrophages by endocytosis and gathered at lysosomes, thus the permeability of lysosome membrane was increased and pH value of lysosome was changed. These results indicated both carbon nanomaterials maybe cause the accumulation of autophagosome through destroy the downstream pathway of autophagy. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/35176]  |
| 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 | 汪子霞. 单壁碳纳米管和氧化石墨烯的免疫毒性效应与机制研究[D]. 北京. 中国科学院研究生院. 2013. |
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