纳米铁酸钴的水生生物和蛋白质毒性机制研究

发布时间：2018-05-02 19:00

  本文选题：纳米毒性 + 纳米铁酸钴　； 参考：《浙江工业大学》2016年博士论文

【摘要】：目前CoFe_2O_4 纳米粒子在医学、环境和工业各方面已获得广泛的应用,使得越来越多的纳米CoFe_2O_4 进入环境,其过量暴露对环境和人体均具有一定风险。因此,急需对CoFe_2O_4 纳米粒子对人体健康和环境生物的潜在影响进行准确的评估。本文以斑马鱼(Daniorerio)和小球藻(Chlorella vulgaris)为生物模型,对纳米CoFe_2O_4 的水生生物毒性进行研究,内容包括纳米CoFe_2O_4 引起的氧化应激、遗传毒性、内分泌干扰效应、纳米CoFe_2O_4 的环境降解、纳米中离子释放以及生物自身对纳米CoFe_2O_4 的抵抗机制等;此外,本文研究了纳米CoFe_2O_4 与牛血清白蛋白(BSA)和酸性磷酸酶(AP)间的相互作用,探究了其热力学数据及蛋白电晕的形成。将斑马鱼胚胎置于环境剂量的纳米CoFe_2O_4 培养液中,分别暴露96和168hpf(hours post fertilization),暴露96和168 hpf,会引起严重的心包水肿、代谢降低、孵化延迟、尾部/脊柱弯曲以及细胞凋亡,且与CoFe_2O_4 呈剂量-效应与时间-效应关系。较低浓度的纳米CoFe_2O_4 会引起过量ROS,进而引起头部、心脏、尾部的细胞凋亡以及生物体内DNA和代谢的变化。暴露168 hpf时,甲状腺激素紊乱、纳米粒子的团聚及粒子释放会导致甲状腺轴的膜损伤、氧化应激及结构损伤。且斑马鱼幼鱼体内T3、T4激素含量升高,导致孵化延迟、眼部、头部畸形等现象。此外,ROS升高会引起8-OHd G DNA聚合物形成,引起DNA损伤,从而产生基因毒性。通过在藻细胞表面的吸附、聚集、释放Fe3+和Co2+以及造成机械损伤,纳米CoFe_2O_4 会损害小球藻细胞形态、膜完整性和通透性。结果也表明纳米CoFe_2O_4 引起的ROS会引起细胞内的氧化应激,导致CAT、GST、AP等抗氧化酶活性下降,并引起遗传突变、代谢及细胞信号传递紊乱。纳米CoFe_2O_4 浓度较低时对ROS含量、CAT、GST活性影响不显著。这项研究表明,诱导ROS产生是CoFe_2O_4 NPs的致毒方式之一,并阐明了在自然环境中可能发生于生物体和纳米颗粒之间的复杂过程。以光谱法作为手段,本文研究纳米粒子与牛血清白蛋白(BSA)、酸性磷酸酶(AP)的相互作用,探讨了纳米CoFe_2O_4 对蛋白质结构和功能的潜在影响作用。结果表明,纳米CoFe_2O_4 通过静态猝灭机制引起BSA和AP的荧光猝灭。负值热力学参数(ΔH和ΔG)说明这种静态猝灭是自发和放热的。ΔS的负值和正值,表明CoFe_2O_4 NPs与BSA和AP间的结合力分别为范德华力、氢键和静电作用。此外,通过TGA、DLS测试证明BSA和AP在CoFe_2O_4 NPs上形成了蛋白质电晕,BSA和AP在CoFe_2O_4 NPs表面的密集包覆,使得负的zeta电位上升。BSA和AP在CoFe_2O_4 NPs上的这种包覆使得磁性饱和值从50.4 emu分别下降到了46.2和45.5 emu。通过对比静态荧光猝灭和理论分析值,进一步分析BSA在CoFe_2O_4 NPs上形成的蛋白质电晕。利用FTIR、UV-CD、紫外可见分光光谱和三维光谱等方法证实CoFe_2O_4 NPs与蛋白质的结合会引起BSA和AP内部微环境改变,引起二级结构和三级结构的改变。此外,同步荧光(SFS)表明CoFe_2O_4 NPs明显改变了BSA和AP内部色氨酸(Trp)残基附近的微环境。通过测定BSA酯酶活性,说明CoFe_2O_4 NPs会引起BSA变性。本文还进一步研究了CoFe_2O_4 NPs对小球藻中AP活性的影响,实验分别测定了CoFe_2O_4 NPs浓度为0和200μM时小球藻液的表观米氏常数(Km)和活化能,表观米氏常数常数分别为0.57和26.5 m M,活化能分别为0.538和3.428 KJ mol-1。-7 Umml-1的表观Vmax值说明酶活性位点完全被NPs占据而没有给酶底物留下空间。结果表明CoFe_2O_4 NPs通过使AP酶展开而降低了酶活性,说明CoFe_2O_4 NPs会通过改变AP酶结构和代谢活性而破坏酶的活性。本文从多个层面阐述了纳米CoFe_2O_4 的生物毒性,为开发环境友好、安全的纳米材料提供了理论性依据。同时,提供了能更好地精确控制和分析纳米颗粒在复杂的生物和环境体系中的方法,为相关监管机构和部门制定和实施严格的法规提供了参考。
[Abstract]:At present, CoFe_2O_4 nanoparticles have been widely used in medical, environmental and industrial aspects, making more and more nanoscale CoFe_2O_4 into the environment, and its excessive exposure to the environment and the human body has a certain risk. Therefore, it is urgent to evaluate the potential effects of CoFe_2O_4 nanoparticles on human health and environmental organisms. The aquatic biological toxicity of nanoscale CoFe_2O_4 was studied with zebrafish (Daniorerio) and Chlorella (Chlorella vulgaris) as biological models, including oxidative stress caused by nano CoFe_2O_4, genetic toxicity, endocrine disrupting effect, environmental degradation of nano CoFe_2O_4, ion release in nanoscale, and biological self on nano CoF. In addition, the interaction between nano CoFe_2O_4 and bovine serum albumin (BSA) and acid phosphatase (AP) was studied, and the thermodynamic data and the formation of protein corona were investigated. The zebrafish embryo was exposed to 96 and 168hpf (hours post fertilization) in the CoFe_2O_4 culture medium of environmental dose. Exposure to 96 and 168 HPF causes severe pericardial edema, metabolic decline, delayed hatching, tail / spinal curvature, and cell apoptosis, with a dose effect and time effect relationship with CoFe_2O_4. The lower concentration of nano CoFe_2O_4 causes excessive ROS, resulting in the apoptosis of the head, heart, tail and DNA and metabolism in the organism. When exposed to 168 HPF, the thyroid hormone disorder, the aggregation of nanoparticles and the release of particles will lead to membrane damage, oxidative stress and structural damage in the thyroid axis. And the increase of T3 and T4 hormone levels in zebrafish young fish causes delayed hatching, eye and head deformities. In addition, the rise of ROS will cause the formation of 8-OHd G DNA polymers. DNA damage, resulting in genotoxicity. By adsorption on the surface of the algae cells, aggregation, release of Fe3+ and Co2+ and causing mechanical damage, nanoscale CoFe_2O_4 can damage the morphology, membrane integrity and permeability of Chlorella, and the results also indicate that the ROS caused by nano CoFe_2O_4 causes oxidative stress in cells, leading to the oxidation of CAT, GST, AP and other antioxidants. The enzyme activity decreased and caused genetic mutation, metabolism and cell signal transmission disorder. When the concentration of nanoscale CoFe_2O_4 was low, the effect of ROS content, CAT, GST activity was not significant. This study showed that inducing ROS production was one of the toxic ways of CoFe_2O_4 NPs, and clarified the possibility of the possible occurrence in the natural environment between the organism and the nanoparticles. The interaction between nano particles and bovine serum albumin (BSA) and acid phosphatase (AP) was studied by spectral method. The potential effects of nano CoFe_2O_4 on the structure and function of protein were investigated. The results showed that the fluorescence quenching of BSA and AP was caused by the static quenching mechanism of nano CoFe_2O_4. The negative thermodynamic parameters were obtained. (delta H and delta G) indicate that the static quenching is spontaneous and exothermic. The negative and positive value of delta S indicates that the binding force between CoFe_2O_4 NPs and BSA and AP is Fan Dehua force, hydrogen bond and electrostatic action respectively. In addition, through TGA, DLS tests show that BSA and AP are formed on the CoFe_2O_4 surfaces to form a protein corona. With the negative zeta potential rising.BSA and AP on CoFe_2O_4 NPs, the magnetic saturation value decreased from 50.4 EMU to 46.2 and 45.5 emu., respectively, by comparing the static fluorescence quenching and theoretical analysis values to further analyze the protein corona formed on BSA in CoFe_2O_4 NPs. Three dimensional spectroscopy and other methods confirm that the combination of CoFe_2O_4 NPs and protein causes the internal microenvironment changes in BSA and AP, and causes the change of the two and three stage structures. In addition, the synchronous fluorescence (SFS) indicates that CoFe_2O_4 NPs obviously changes the microenvironment near the tryptophan (Trp) residues in BSA and AP. The effect of CoFe_2O_4 NPs on the activity of AP in Chlorella was further studied. The apparent Michaelis constant (Km) and activation energy of Chlorella in the concentration of CoFe_2O_4 NPs at 0 and 200 u M were further investigated. The apparent Michaelis constant constant was 0.57 and 26.5 m M respectively, and the activation energy was 0.538 and 3.428 KJ respectively. The apparent Vmax value of mml-1 indicates that the enzyme active site is occupied by NPs and does not leave space for the enzyme substrate. The results show that CoFe_2O_4 NPs reduces the activity of the enzyme through the expansion of the AP enzyme, indicating that CoFe_2O_4 NPs will destroy the activity of the enzyme by changing the structure and metabolic activity of the AP enzyme. This article expounds the birth of nano CoFe_2O_4 from several levels. It provides a theoretical basis for the development of environmentally friendly and safe nanomaterials. At the same time, it provides a better way to accurately control and analyze nanoparticles in a complex biological and environmental system, and provides a reference for the relevant regulatory bodies and departments to formulate and implement strict regulations.

【学位授予单位】：浙江工业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ138.12;TB383.1
，

本文编号：1835093