| 题名 | 纳米银表面修饰对其生物学效应的调控作用及分子机制研究 |
| 作者 | 龙艳敏 |
| 学位类别 | 博士后 |
| 答辩日期 | 2015-04 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 江桂斌 ; 周群芳 |
| 关键词 | 纳米银，表面修饰，氧化应激，接触体系，生物转化，nanosilver, surface modification, oxidative stress, kallikrein-kinin system, biotransformation |
| 学位专业 | 纳米毒理 |
| 中文摘要 | 由于纳米银优异的抗菌活性以及对细菌不易产生耐药性，因而目前已被作为抗菌有效成分广泛应用于生物医药、能源设备、家用电器、化妆品与食品等多个方面。据统计，截止2014年，涉及纳米银的商品已达400多种，高居各种纳米制品之首。现有研究显示一些含纳米银的商品例如织物等在正常使用及洗涤过程中，将释放银离子或纳米颗粒进入环境，从而引起了人们对于这类纳米材料生态、环境及生物毒性的极大关注。目前虽有不少关于纳米银毒理学效应的研究报道，但基于纳米银本身材料结构的复杂性，以及环境与生物体系的不确定性，在纳米银毒性机制阐释中仍存在很多新问题、新方法丞待探索研究。 纳米银表面的Ag0在环境或生体系中能自发地氧化溶解释放出Ag+，同时O2不完全还原生成活性氧化物（ROS），进而对生物体产生相应的毒性效应，因此关于纳米银的致毒物种及相应的致毒途径与机制非常复杂，至今尚无明确定论，针对纳米银致毒机制的研究一直是纳米毒理领域的重要研究课题。另外，同其他纳米材料一样，纳米银表面结构在很大程度上影响了其物化特性，也影响了其生物活性。近来相关报道指出，纳米银生物毒性呈现出显著的表面结构依赖特征，因此，阐明表面修饰调控的纳米银生物学效应具有重要意义。我们首先合成不同表面修饰的纳米银，通过比较这些纳米银颗粒的生物活性或毒性差异，阐明表面修饰对纳米银生物效应的影响，从而探讨了包括致毒物种及致毒途径、方式在内的纳米银致毒机制。 (1) 利用配体交换法制备得到化学结构上系统可比且配位作用明确的小分子修饰的纳米银颗粒（AgNPs），通过研究它们毒性相关的关键性质（Ag+释放和ROS产生），及对大肠杆菌E. coli生物毒性的对应关系，得出AgNPs的致毒物种为其释放的Ag+而非表面同时产生的ROS。加入还原剂GSH或Ag+络合剂Na2S2O3时，纳米银的抗菌活性基本完全抑制，提示纳米银是通过释放的Ag+使细菌本身产生ROS导致了最终的抗菌活性。进一步结合SEM表征、胞内ROS和Ag定量及氧化应激指标（脂质过氧化）的检测，我们提出了纳米银的抗菌活性机制为：纳米银释放的Ag+，破坏细菌膜结构，扰乱膜上呼吸链的正常功能，导致胞内ROS的积累，最终产生氧化应激，引起杀菌效应。 (2) 血管舒缓素-激肽系统（KKS）是人体血浆中重要的生物酶体系，调控了血压、炎症、凝血等一系列生物学功能。根据经典理论，KKS可在内源或外源负电荷表面激活，从而引起包括血管通透性在内的许多生理功能的改变。本研究发现，与高岭土类似，表面带负电荷的纳米银可作为外源纳米级活化剂，快速且剂量依赖性地激活离体血浆中的KKS体系，而且这种激活效应为颗粒效应，相当剂量的银离子或表面修饰小分子本身并不能引起KKS活化。圆二色谱及western blot表征也证实了负电荷纳米银对KKS体系中重要的蛋白酶原FXII、PPK、HK的顺序激活。以C57/B6小鼠为活体实验模型，通过颈静脉注射不同剂量纳米银时，也同样观察到了活体血浆KKS体系的明显激活，与离体体系不同的是，活体存在明显的调控机制，当循环时间进一步延长时，被激活活性成分血管舒缓素（PK）的含量出现了先上升后下降的趋势。另外，小鼠眼玻璃体内注射纳米银可引起其视网膜血管通透性的明显增加，血浆蛋白PPK外渗，染料Evans blue积累等活体生物学效应。以组成血-视网膜屏障（BRB）的主要成分视网膜内皮细胞为模型，结合单层细胞通透性transwell检测，细胞免疫荧光和western blot分析，得出纳米银增加视网膜血管通透性机制为：KKS体系被负电荷纳米银激活后，产生的下游炎性调节因子血管舒缓激肽（BK）与视网膜内皮细胞表面受体B2结合，从而诱导细胞间粘合连接蛋白VE-cadherin的磷酸化脱落，胞间连接打开，BRB破坏，视网膜血管通透性增加。 (3) 在比较Ag+和纳米银对KKS体系激活效应的实验中，我们发现高剂量Ag+可对KKS系统产生与负电荷纳米银相似的激活效应。利用SEM和STEM电子显微术，对高剂量Ag+处理后的血浆样品进行形貌及成分分析发现，血浆中有纳米银颗粒的生成。基于体外模拟体系，我们进一步证实GSH和还原型辅酶II（NADPH）是血浆中Ag+还原为纳米银的关键还原剂。结合热力学氧化还原电势的计算结果，可以发现血浆中Ag+转化为纳米银的生物学过程中GSH、NADPH起到还原作用，该研究结果对于阐释纳米银或Ag+的生物毒性具有重要启示作用。 |
| 英文摘要 | Due to broad-spectrum antibacterial activity and little bacterial drug resistance, nanosilver (nAg) has been extensively applied in multiple fields including biomedicine, energy, household appliance, cosmetics and food. By 2014, the catalogue of commercial products containing nAg has exceeded 400 types, which tops the list of the nanomaterial-contained products. Recent work has reported that nAg-contained fabrics can release most of Ag in both forms of Ag ion and nanoparticle. Therefore, a big concern has been raised on the adverse effects of nAg bringing to the ecology, environment and organism. Even though some related toxicological studies have been performed, there are many tough problems which need to be addressed in-depth due to the complicated structure of nAg and diversity of environment and organism. Ag0 on the surface of nAg can be spontaneously oxidized, dissolved and release Ag+ into the ambient environment or biological system. At the same time, the reactive oxygen species (ROS) are formed via the incomplete reduction of O2. These two active species can lead to distinct toxicities. The actual effective species and the related toxicity mechanisms are thus complex and still remain open questions. Our research has focused on the studies for the mechanisms of nanosilver induced toxicology including active species. The effects of diverse surface coatings on the toxicities of nanosilver are dicussed. 1) A series of Ag nanoparticles (AgNPs) coated with diverse organic molecules have been synthesized through exchanging the ligands of precursor, citrate-capped AgNPs. According to the properties of Ag+ release and ROS production, and the corresponding toxicities of diverse AgNPs to E. coli, it is concluded that Ag+ is the main active species for AgNPs induced antibacterial activity. The addition of antioxidant GSH or ligand Na2S2O3 caused the complete inhibition in AgNPs caused effect, indicating that the intracellular ROS was induced by released Ag+ from AgNPs. SEM characterizations, intracellular ROS and Ag level quantification, and the evaluation of oxidative stress markers (8-OH dG generation and lipid peroxidation) confirmed the mechanism that AgNPs were firstly oxidized and released Ag+, which subsequently damaged cell membrane structure and interrupted the respiratory chain to provoke intracellular ROS, thus causing oxidative damge to the bacteria. 2) Kallkrein-kinin system (KKS), an important plasma enzymatic system, can be activated by negatively-charged surfaces (glass and kaolin), causing some disorders like increased vascular permeability. Our work found that negatively-charged AgNPs also can be the potential activator to active the KKS in ex-vivo plasma in a time and dose dependent manner. The characterizations of circular dichroism spectrum and western blot assay on the purified protein system verified the cascade activation of zymogen FXII, PPK and HK. Jugular injection of AgNPs caused the in-vivo plasma KKS activation, thus leading to the increased retinal vascular permeability evidenced by Evan blue assay and dextran-FITC image assay. Elevation of retinal PPK level also confirmed the plasma protein leakage from the increased vascular permeability. The studies on human retinal endothelial cells clarified the mechanisms for the phenotype observed above. Taken together, the activation of KKS cascade by negatively charged AgNPs liberates the biological active peptide, BK, which binds with B2 receptor and results in the shedding of VE-cadherin. The loss of VE-cadherin impairs AJs integrity in retinal endothelial cells of the vascular wall which causes increased retinal vascular permeability. 3) When the effects of Ag+ and AgNPs on KKS activation were evaluated, we found that high level of Ag+ (400 ppm) could induce the similar effect as what AgNPs caused. As expected, the generation of AgNPs in Ag+ incubated plasma sample has been witnessed by the electron spectroscopic characterizations. Furthermore, according to the studies on purified peptide GSH and coenzyme II NADPH, a mechanism that GSH and NADPH can reduce the Ag+ into Ag0 has been proposed for the formation of nanoparticulate Ag. Theoretically, the possibilities have also been validated in the terms of reduction capability referring to thermodynamic redox potentials. This work provides important hints on nAg toxicology. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/34350]  |
| 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 | 龙艳敏. 纳米银表面修饰对其生物学效应的调控作用及分子机制研究[D]. 北京. 中国科学院研究生院. 2015. |
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