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体外实验研究不同粒径纳米银的氧化应激效应
Study the Oxidative Stress of nanoAg with Different Diameters in Vitro
【作者】 张斌;
【导师】 刘汝涛;
【作者基本信息】 山东大学 , 环境工程(专业学位), 2015, 硕士
【摘要】 纳米科学技术历经了几十年的探索和发展已成为当前最为活跃的研究领域,是世界各国研究的热点。纳米银(nanoAg)由于具有独特的电子,光学,催化,机械和抗菌等优良性质使得其在研究和应用上大量使用,是应用最广泛的纳米材料,与此同时也造成了其对相关工作人员和局部环境的较大剂量暴露。关于nanoAg的毒性作用的研究已有陆续报道,但是nanoAg对蛋白质等功能生物大分子的毒性作用以及细胞毒性的机制尚未阐明。为了保证nanoAg在纳米材料领域的发展,减少nanoAg使用过程中对人类及生态环境的危害,急需对nanoAg的毒性效应做全面、深入的研究。研究表明氧化应激几乎与所有的疾病有关,环境污染物对生物体产生的危害与氧化应激密切相关。在氧化应激过程中,活性氧类物质(ROS)在生物体内大量表达,参与并促进多种疾病的发生发展过程,造成DNA/RNA、蛋白质、脂类等生物分子的氧化损伤,从而导致机体代谢紊乱,诱发疾病。因此,利用氧化应激这一生物学效应指标来评价环境污染物的毒性效应,有助于人们深入了解环境污染物的致病机理,全面评价其毒性,为疾病的早期诊断、预防和治疗提供科学依据。本文以机体氧化应激理论为基础,选择机体内与氧化应激密切相关的过氧化氢酶(CAT)及超氧化物歧化酶(SOD)为靶,从分子水平及细胞水平上探讨了nanoAg对其结构和功能的影响。本文包括以下两个部分:第一部分:第一节:利用化学还原法制得粒径在30 nm-50 nm之间的nanoAg颗粒;利用荧光光谱、吸收光谱、圆二色光谱、共振光散射光谱等光谱学技术以及透射电镜等现代分析测试技术,从分子水平上探讨了nanoAg对CAT的毒性作用模式和剂量-效应关系。NanoAg可导致CAT发生荧光猝灭,且属于动态猝灭;nanoAg能够明显改变CAT分子结构:使CAT分子α-螺旋含量降低,β-折叠含量增加,骨架结构变得松散,共振光散射实验的研究结果表明CAT与nanoAg作用后可以使nanoAg粒子粒径分布更加均匀,并且粒径变小,并且得到了透射电镜实验的进一步证实;CAT结构的改变最终导致了其活性升高。第二节:利用购买的粒径为20nnm左右的nanoAg,使用荧光光谱、紫外-可见吸收光谱、圆二色谱等多种光谱学技术探讨了nanoAg与SOD的作用模式,以及由此导致的SOD的功能变化。NanoAg可导致SOD荧光猝灭,荧光寿命及共振光散射实验证明该过程同时存在动态猝灭和静态猝灭;紫外-可见吸收光谱、同步荧光光谱以及圆二色谱的研究结果证实了nanoAg与SOD作用后,影响了其分子结构,从而使SOD的酶活性略有下降。第二部分:对比两种粒径nanoAg(20nm、60nm)在细胞水平上的毒性。以C57BL/6J系小鼠原代肝细胞为研究对象,通过CCK-8试验为对比研究了nanoAg暴露剂量对原代小鼠肝细胞的活力效应;以原代小鼠肝细胞为研究对象,对其进行体外染毒,测定不同nanoAg剂量下丙二醛(MDA)含量、CAT活性。研究发现nanoAg可以诱发原代小鼠肝细胞产生氧化应激,且粒径小的nanoAg比粒径大的nanoAg毒性更大。本研究表明,nanoAg可以影响氧化应激关键酶(CAT和SOD)的结构及其功能,并且诱导细胞产生氧化应激,显示出一定的分子毒性和细胞毒性,进而影响生物体的正常生理功能,且在细胞水平上粒径小的较粒径大的nanoAg毒性更大。
【Abstract】 Nano-technology has tremendous potential applications in biology and medicine, including disease diagnosis, drug targeting transmission, and biological sensors. Nano-silver (nanoAg) is developed into a new nano-material based on nano technology. Because of its unique characteristics, nanoAg has a wide range of applications in detection, printed electronics, disinfection and food storage. Furthermore, NanoAg materials have a close relationship with modern pharmacology and medicine. NanoAg has more qualitative bactericidal capacity and could overcome drug resistance. All mentioned above results in the relevant staff and local environmental larger dose exposure.Humans have attached great attention to its environmental hazards caused by the gradually increasing use of nanoAg. So far at home and abroad, the previous methods investigating the toxicity of nano-silver mainly focused on morphology, determination of mitochondrial function, cell proliferation, enzyme activity and cytotoxic testing. The overall level of toxicity testing has also been reported. However, there is a lack of research on the mechanism at the molecular level.In order to ensure the positive development of nano-silver materials, and reduce its injury to the natural ecological system, especially human beings, there is an urgent need to do a comprehensive and in-depth study on the toxic effects of nano-silver.This thesis includes the following three sections.In the first section the toxic effects of nanoAg on catalase were thoroughly investigated using steady state and time resolved fluorescence quench-ing measurements, ultraviolet-visible absorption spectroscopy, resonance light scattering spectroscopy (RLS), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM). NanoAg could decrease the amount of alpha-helix and increase the beta sheet stucture, leading to loose the skeleton structure of catalase. The characteristic fluorescence of catalase was obviously quenched, which showed the exposal of internal hydrophobic amino acids enhanced, and its quenching type is dynamic quenching. The result of RLS and TEM showed that the distribution and size of nanoAg become more uniform and smaller after their interaction, resulting in a decrease of RLS intensity. NanoAg could make the activity of catalase rise. By changing the structure of catalase, nanoAg increases its enzymatic activity to a certain extent, breaking down its balance in vivo, thereby affecting the normal physiological activities. NanoAg has obvious toxic effects on catalase.In the second section the effect of exposure to nanoAg on the structure of superoxide dismutase (SOD) was thoroughly investigated by fluorescence measurements, synchronous fluorescence spectroscopy, steady state and time resolved fluorescence quenching measurements, ultraviolet-visible absorption spectroscopy (UV), resonance light scattering (RLS), circular dichroism spectrum (CD), isothermal titration calorimetry (ITC) and high-resolution transmission electron microscopy (HRTEM). Through van der Waals force, nanoAg interacted with Cu-Zn SOD and influenced the active site by inducing structural changes which influence the function of SOD. The fluorescence studies show that both static and dynamic quenching processes occur.In the third section, we made a comparison of nanoAg toxicity in two diameters at the cellular level. C57BL/6J mice primary hepatocytes used for the study, we made the study of CCK-8 means exposured from nanoAg on primary mouse hepatocytes; We measured malondialdehyde (MDA), SOD activity, CAT activity at different doses of nanoAg. The experimental results showed nanoAg could induce oxidative stress and nanoAg with smaller diameter express more toxicity compared with bigger ones.The research results show that, nanoAg can affect the structure of a key enzyme as well as its function, and induce oxidative stress. It also shows a certain toxicity to molecules and cells, thereby affecting the normal physiological function of the organism.
【Key words】 NanoAg; Catlase; Superoxide dismutase; Oxidative stress; Toxicity mechanism;