【作者】 李平；

【导师】 何德富；

【作者基本信息】 华东师范大学 ， 环境科学， 2017， 硕士

【摘要】 因其优良的特性,纳米材料近年来在生物医学、电子信息、环境保护、能源和国家安全等方面有了空前广阔的应用。以石墨烯(graphene,G)和氧化石墨烯(graphene oxide,GO)为代表的石墨烯类是应用最为广泛的纳米材料。另外,金属类纳米银(Ag-NPs)和纳米氧化锌颗粒(ZnO-NPs)近年来大量运用于新材料和医疗新技术方面。纳米材料的生产、使用和处置将最终导致其流入空气、水或土壤等环境介质和生物体内,威胁生态安全,其生物毒性和对人类健康的潜在威胁备受关注。已有研究显示多种纳米材料具有生殖毒性和发育毒性等生物效应,但研究尚不全面和系统,特别是慢性神经毒性的资料缺乏。本研究则选取秀丽线虫(C.elegans)为模式生物,利用其生命周期短和神经系统相对简单等优势。选取石墨烯、氧化石墨烯、石墨烯量子点、纳米银和纳米氧化锌五种常见纳米材料,详细表征了理化性质。不同浓度悬浮液暴露于秀丽线虫6天后检测其致死浓度水平。并进一步使用亚致死浓度慢性暴露,重点研究五类纳米材料对秀丽线虫运动行为影响和神经毒性。通过荧光标定的转基因线虫详细探讨和比较了这些纳米材料对多巴胺能、谷氨酸能和GABA能神经元的毒性效应。主要结果为:1、G 和 GO 成片状,厚约 0.8-1.2nm;GQDs、Ag-NPs 和 ZnO-NPs 均为颗粒,粒径在30-50nm。悬浮液暴露6d结果显示,低于100mg/L的G和GQDs对线虫没有明显致死效应,而50-100mg/LGO表现出明显致死效应,其半致死浓度(LC50)为51.22 mg/L。而金属类Ag-NPs和ZnO-NPs对线虫有较强致死效应,其 LC50 分别为 2.723mg/L 和 1.852mg/L。2、从石墨烯类观察结果来看,暴露后纳米材料在线虫体内有广泛的累积和分布。G和GO暴露后主要分布于线虫的咽腔及肠腔内;而荧光显微观察显示GQDs能剂量依赖型分布到线虫身体全身多器官。五种纳米材料慢性暴露对秀丽线虫的生长具有不同程度的抑制作用,表现为身体变短变细。3、五种纳米材料慢性暴露对秀丽线虫的运动行为具有抑制作用,随暴露浓度增大而抑制作用加强。暴露后线虫头部摆动频率、身体弯曲频率和咽泵运动频率变慢,反映其身体局部运动受损。进一步通过分析动物爬行行为,发现暴露后爬行轨迹弯曲不规则,爬行距离缩短,速度变慢,弯曲振幅变小和爬行过程角度方向变化趋缓,显示身体僵硬、不灵活和平衡能力降低等损失特征。4、基于荧光标定的转基因线虫研究显示,三种石墨烯类纳米材料高浓度(50-100mg/L)暴露能抑制da::GFP 和 eat-4::GFP 的表达,但unc-::GFP表达没有变化。同时多巴胺能和谷氨酸能神经元出现胞体萎缩变形、树突断裂等损伤形态。金属类Ag-NPs和ZnO-NPs暴露则导致da-1::GFP和unc-4::GFP表达的荧光强度显著下降;但同样浓度暴露对eat-4::GFP表达没有明显影响。结果反映这两种金属类纳米材料可易感性损伤多巴胺能神经元和GABA能神经元。以上结果表明:1、五种常见的纳米材料对秀丽线虫均具有慢性毒性。从LC50和生长抑制效应的对比上看,两种金属类的毒性比石墨烯类强,其毒性强弱次序为:ZnO-NPs>Ag-NPs>GO>G>GQDs。2、五种纳米材料的亚致死浓度慢性暴露具有不同程度的神经毒性。慢性毒性表现在动物运动行为能力的下降和相关神经元的损伤。运动行为的毒性表明动物出现身体僵硬、运动不能和平衡丧失等特征。另外发现金属类和石墨烯类纳米材料对多巴胺能神经元、GABA能神经元和谷氨酸能神经元的损伤具有不同的易感性,其详细机制尚需深入研究。本文基于秀丽线虫的系统研究充分揭示了常见纳米材料长期接触诱发行为缺陷和神经损伤的毒性,并存在健康风险。在纳米材料越来越多地运用到日常生活的情况下,本文研究结果为纳米材料的风险评价、安全管理和标准制定等提供了重要参考。本文工作也为深入探究纳米材料的毒性机制及其预防措施奠定了基础。更多还原

【Abstract】 Nanomaterials have unique properties,and are extensively used in fields of biomedicine,telecommunication,environmental protection,energy,national security in recent years.Among nanomaterials,graphene(G)and graphene oxide(GO)are most common kinds;silver nanoparticles(Ag-NPs)and zinc oxide nanoparticles(ZnO-NPs)are recently applied in new materials and medical technology.In the process of production,utilization and disposal,fragments of nanomaterials will occur in the air,water,soil and organisms.Nanomaterials will probably threaten the ecological safety,and its bio-toxicity and the potential risks to human health receive much concern.Biological effects including reproductive toxicity and developmental toxicity of some nanomaterials have been revealed in previous studies.However,their impacts on environment and health are still not well understood,especially about risks of chronic neurotoxicity.In this study,Caenorhabditis elegans was selected as a model organism,due to the advantages of a short life cycle and a simple nervous system.Five kinds of commonly used nanomaterials,graphene,graphene oxide,graphene quantum dots(GQDs),Ag-NPs and ZnO-NPs were studied in detail.After phy-chemical properties of these nanomaterials were characterized,the lethal concentrations were measured for 6 d exposure of suspension liquids in C.elegans.The effects of chronic exposure to these nanomaterials on locomotor behaviors of nematodes and their neurotoxicity were fully investigated under sub-lethal exposure conditions.The toxic effects of these nanomaterials on dopaminergic,glutamate and GABAergic neurons were compared using fluorescence-labeled transgenic nematodes.Main results include that::(1)G and GO are in the form of flakes,with a thickness of 0.8-1.2 nm.GQDs,Ag-NPs and ZnO-NPs are in the form of granules with diameters of 30-50 nm.After chronically exposed to less than 100mg/L G and GQDs for 6 d,C.elegans showed no lethal effects;while 50-100mg/L GO resulted in significant lethality,the median lethal concentration(LC50)was 51.22 mg/L.However,Ag-NPs and ZnO-NPs had strong lethal effects on nematodes;LC50 was 2.723 mg/L and 1.852 mg/L,respectively.(2)After exposure,graphene and GO nanomaterials had extensive accumulated and distributed in the body of nematodes.For example,G and GO nanosheets were primarily distributed in pharynx and intestine;while GQDs were widely distributed in various organs of nematodes in dose-dependent manners.Chronic exposure to five nanomaterials caused inhibitory effects on the growth of the nematodes,such as shorter body than the controls.(3)Chronic exposure to five nanomaterials caused inhibitory effects on the movement behavior of the nematodes in concentration dependent manners.Nanomaterials resulted in significant declines in the frequency of head thrashing,body bending and pharyngeal pumping,which indicated the damage of locomotor behaviors.Furthermore,we found that crawling tracks were irregularly curved,crawling distances were shortened,amplitude of bending wave became smaller and the crawling angles changed slower in the exposed nematodes in comparison with the controls.These results indicated that nematodes presented characteristics of movement impairment including bradykinesia,hypokinesia and unbalance and so on.(4)Using transgenic nematodes,results showed that high concentrations(50-100mg/L)of three kinds of graphene-based nanomaterials could inhibit the protein expression of dat-1∷GFP and eat-4::GFP,but unc-47::GFP expression did not change.Morphology results showed deformation of the cell body,dendritic fracture and other damages in dopaminergic and glutamate neurons.Exposure of Ag-NPs and ZnO-NPs resulted in a significant decrease in the fluorescence intensity of dat-1::GFP and unc-47::GFP,but no changes in the expression of eat-4∷ GFP.The results indicated that these metal nanomaterials were susceptible to dopaminergic neurons and GABAergic neurons.These results indicate that:(1)Five kinds of commonly used nanomaterials possess chronic toxicity to Caenorhabditis elegans.To comparison with LC50 and growth inhibition effects,toxicity of two metal nanoparticles is stronger than that of graphene-based nanomaterials.The order of toxicity is:ZnO-NPs>Ag-NPs>GO>G>GQDs.(2)Chronic exposure to sub-lethal concentrations of these nanomaterials causes neurotoxicity with different degrees.Chronic neurotoxicity includes behavioral deficits and neural damages in C.elegans.Behavioral toxicity shows impairment character such as bradykinesia,hypokinesia and unbalance and so on.In addition,metal and graphene-based nanomaterials have different susceptibility to dopaminergic neurons,GABAergic neurons and glutamate neurons,and detailed mechanisms need to be further studiedIn the present study,we have fully revealed the health risk of commonly used nanomaterials,behavioral deficits and neurotoxicity induced.In recent year,nanomaterials have been increasing used in daily life.These findings provide important references for risk evaluation,safety management and standard establishment of nanomaterials.This study also builds the foundation for the further study on the toxicity mechanism and preventive measures of nanomaterials.更多还原