| 题名 | 莱茵衣藻对纳米银的生物吸收与转化及 响应机理研究 |
| 作者 | 王松山 |
| 学位类别 | 博士 |
| 答辩日期 | 2016-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张淑贞 |
| 关键词 | 纳米银，颗粒吸收，生物转化，缺铜反应，转录因子 CRR1 silver nanoparticles, cellular internalization, biotransformation, copper deficiency response, transcription factor CRR1 |
| 其他题名 | Internalization，biotransformation and biological effects of silver nanoparticles in Chlamydomonas Reinhardtii and the related mechanisms |
| 学位专业 | 环境科学 |
| 中文摘要 | 纳米银因具有广谱抗菌活性而在工业生产、日常生活用品和医药卫生中广泛应用，这些产品在其生产和使用过程中存在纳米银的释放风险，导致进入环境中纳米银数量不断增加，并引起人们对纳米银生物安全性和潜在毒性效应的关注，纳米银的环境行为和毒性效应及其机制也因此成为环境科学研究的热点之一。本研究以单细胞莱茵衣藻为模式生物，综合利用多种先进表征技术和分子生物学方法，围绕纳米银的生物吸收与转化、适应性响应以及相关分子机制开展了如下几个方面研究： 利用液相还原成核和光照诱导颗粒组装的方法制备了粒径 20 nm左右的聚乙烯吡咯烷酮（PVP）修饰球形纳米银颗粒，其在暴露介质中稳定性高，不易发生团聚沉淀。在此基础之上综合利用纳米级二次离子质谱（NanoSIMS）、同步辐射X射线吸收光谱（XAS）和高角环形暗场扫描透射电镜技术（HAADF-STEM），并结合选区电子衍射和能谱分析，研究了暴露纳米银后莱茵衣藻细胞内银的分布和形貌特征以及赋存形态。NanoSIMS和 HAADF-STEM微区化学成像表明暴露纳米银后衣藻细胞壁膜间隙和细胞质区室中均存在明显的银富集。选区电子衍射晶体结构分析、能谱元素组成及 XAS形态分析证明壁膜间隙中富集的银为直径10-20 nm的纳米银颗粒，而在银离子暴露下则未观察到纳米银颗粒。在细胞质区室中银的分布和形态与银离子暴露相同，主要以 β-硫化银晶体和银巯基络合物形态存在。上述结果首次证明了纳米银颗粒能够直接进入莱茵衣藻细胞壁膜间隙，而细胞质内的银则是胞质外纳米银解离释放出银离子的吸收、硫化和区室化隔离所致。本研究通过综合利用先进谱学技术阐述了纳米银进入衣藻细胞的途径及其在胞内的转化与区室化机制，同时也为研究其他纳米材料的生物过程提供了可借鉴的研究方法。 为阐明纳米银进入衣藻细胞和随后生物转化的分子机理，以及纳米银胁迫的生物适应性响应机制，本研究利用转录组测序技术分析了衣藻暴露无毒性效应剂量纳米银和银离子后细胞转录组的变化。结果表明纳米银和银离子暴露后编码细胞壁和鞭毛功能性组成蛋白的转录本明显降低，导致细胞壁疏松甚至破损，鞭毛消失。细胞壁结构的破坏也为纳米银颗粒穿透提供了有效途径。另一方面，银离子暴露时细胞内与硫代谢相关的部分基因表达量发生显著改变，而纳米银暴露时则无显著变化，这与我们在前面研究中观察到的纳米银以释放银离子方式在细胞质内硫化的过程相符。其中 CDO2和 RDP2等与有机态硫重复利用相关的基因出现上调，而硫酸根胞外吸收转运和叶绿体内同化还原生成巯基化合物（半胱氨酸、谷胱甘肽和植物络合素等）相关的基因并无显著变化，但半胱氨酸向蛋氨酸转化过程相关酶的编码基因出现下调。硫代谢过程的变化与细胞内银硫化过程的关系还需要进一步验证。此外，研究中发现纳米银和银离子暴露均可引起显著的莱茵衣藻细胞功能性缺铜和氧化应激反应。纳米银暴露时缺铜响应基因上调的持续时间较银离子暴露长，而银离子暴露引起的氧化应激较纳米银暴露更为明显。缺铜反应激活 CYC6和FDX5等一系列相关缺铜响应基因的表达，导致细胞内铜的过量累积。MSD3和 TRX类基因的显著变化分别与叶绿体和细胞质内的氧化应激反应有关，并且 MSD3上调幅度高达 1000倍以上，可以作为氧化应激反应的指示性基因。 CRR1是衣藻细胞内缺铜响应基因的转录因子之一，其调控基因包括CYC6和 FDX5等。为明确缺铜反应在衣藻适应纳米银胁迫过程中的作用，我们获得了CRR1编码基因单碱基缺失突变株（crr1突变株）和回复株，以便与野生型进行对比实验。结果证明转录因子 CRR1缺陷可导致衣藻细胞在纳米银暴露条件下无法正常激活 CYC6和 FDX5等缺铜响应基因的表达，同时不会表现出细胞内铜的过量累积。转录因子 CRR1缺陷时纳米银在衣藻细胞中的累积及其半数抑制浓度并无显著变化，但是纳米银向银离子转化过程受到抑制，野生型衣藻细胞中一价银含量显著高于 crr1突变株。MSD3与缺铜响应基因表达趋势分析以及银离子毒性测试均证明莱茵衣藻细胞通过招募转录因子 CRR1调控的缺铜响应基因的表达以缓解纳米银释放出银离子对细胞产生的稳态破坏。本研究首次证实衣藻细胞铜稳态紊乱与纳米银生物转化和毒性效应的关系，为进一步阐明纳米银与生物相互作用的机理奠定基础。 |
| 英文摘要 | Silver nanoparticles (AgNPs) are widely used in numerous technologies and incorporated into a wide array of consumer products due to their antibacterial properties. AgNPs embedded in the consumer products are expected to make their way during manufacture, usage or disposal into aquatic environment and the amount is expected to continue increasing. The released AgNPs will inevitably interact with aquatic organisms, spurring growing attentions on the potential risk of AgNPs to the environmen. Therefore, there is a considerable effort underway to understand the environmental behaviors and toxicities of AgNPs in aquatic environments. In this study, a combination of high-resolution techniques and molecular biological methods was employed to investigate the cellular internalization and intracellular biotransformation of AgNPs in the model organism Chlamydomonas reinhardtii, and the adaptive responses and related mechanism in the algal cell under AgNP stress. AgNPs coated with PVP in the diameter of～20 nm were synthesized by liquid reduction together with light-induced self-assembly method, which were stable enough to keep well dispersed in the exposure medium. Then a combination of high resolution imaging and in situ detection spectroscopic techniques was employed to systematically investigate the intracellular localization, morphology and chemical speciation of silver in the cells of Chlamydomonas reinhardtii, a unicellular freshwater green alga, after exposure to AgNPs. High resolution secondary ion mass spectrometry (NanoSIMS) and high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) together with energy dispersive spectroscopy (EDS) and selected area electron diffraction (SAED) collectively confirmed that AgNPs entered the periplasmic space after cellular internalization into the algal cells. Silver was also found to coexist with sulfur inside the cytoplasm in both crystalline and amorphous forms, which were further identified as β-Ag2S and silver thiolates with synchrotron X-ray absorption spectroscopy (XAS). In combination, these analyses demonstrated that silver inside algae was attributed to the uptake and sequestration of Ag+ ion released from AgNPs, which was further sequestrated into cellular compartments. This study provides solid evidence for particle internalization and biotransformation of AgNPs after interaction with algae, and also a valuable reference for understanding the interactions of nanoparticles with cells and bio-macromolecules. To understand the molecular mechanisms that underlie the internalization, biotransformation of AgNPs, and adaptive responses to AgNPs in algal cells, the transcriptome of algae exposed to non-toxic concentrations of AgNPs and Ag+ ion were monitored by RNA-seq. There was an obvious decrease of transcripts encoding proteins that functioned in the cell wall and flagella, engendering the loss of flagella and less tight and even broken of cell walls. The increase of permeability of cell wall was conducive to the direct penetration of AgNPs. Genes involved in sulfur metabolism were not significantly altered after exposure to AgNPs, whereas some were obviously changed for the exposure to Ag ion. This was in line with the observation that sulfidation of Ag+ ion dissolved from AgNPs after entering the cytoplasm. CDO2 and RDP2, relating to the recycle of sulfur in organic molecules,were upregulated for the exposure to Ag ion. Genes involved in the uptake and translocation of sulfate and synthesis of cysteine, glutathione and phytochelatins from sulfate were in the same level with the control, while those encoding enzymes that catalyzed the synthesis of methionine from cysteine were down regulated. Further studies are necessary to investigate the relationship between the change of sulfur metabolism and sulfidation of Ag. Moreover, both exposures of AgNPs and Ag+ ion induced copper deficiency response and oxidative stress in the algal cells. The upregulations of copper deficiency responsive genes sustained a relative longer time for the exposure to AgNPs than that of Ag+ ion, while the oxidative stress was more obvious for the exposure to Ag+ ion than that of AgNPs. Copper deficiency activated the upregulations of a set of relative genes including CYC6 and FDX5 and et al, resulting in an excessive accumulation of copper in the algal cells. The changes of MSD3 and TRX indicated the oxidative stress in the chloroplast and cytosol. The fold change of MSD3 reached up to 1000, and therefore it was considered to be one sentinel gene to indicate oxidative stress. CRR1 is one of the transcription factors that regulate the expressions of CYC6 and FDX5 and et al. To elucidate the role of copper deficiency response in the adaptive responses to AgNPs stress, the crr1 mutant with complete deficient of CRR1 due to single nucleotide deletion of the encoding regions, and the complemented strain were obtained to make a comparative study with the wild type. The results indicated that defect of transcription factor CRR1 made the algal cell incompetent to activate the expressions of copper deficiency responsive genes such as CYC6 and FDX5, and no excessive accumulation of copper in the algal cell. The defect of CRR1 did not affect the accumulation of Ag in the algal cell and EC50 value of AgNPs, while it reduced the transformation of AgNPs to Ag ion, indicating the higher accumulation amount of monovalent Ag in the wild type than that in the crr1 mutant. The trends of expressions of MSD3 and copper deficiency responsive genes and the Ag ion toxicity assay confirmed that copper deficiency responsive genes were responsible for maintaining homeostasis on perturbations caused by Ag ion and function in algal resistance to the toxic effects of Ag ion released from AgNPs. This study confirmed the relationship between the disruption of copper homeostasis and biotransformation and toxic effects of AgNPs in algal cells, which was conduicive to advance the understand of mechanisms of AgNP-biological interactions. |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.rcees.ac.cn/handle/311016/37021]  |
| 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 | 王松山. 莱茵衣藻对纳米银的生物吸收与转化及 响应机理研究[D]. 北京. 中国科学院研究生院. 2016. |
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