發(fā)布時間：2018-05-02 19:00

  本文選題：納米毒性 + 納米鐵酸鈷�。� 參考：《浙江工業(yè)大學》2016年博士論文

【摘要】：目前CoFe_2O_4 納米粒子在醫(yī)學、環(huán)境和工業(yè)各方面已獲得廣泛的應用,使得越來越多的納米CoFe_2O_4 進入環(huán)境,其過量暴露對環(huán)境和人體均具有一定風險。因此,急需對CoFe_2O_4 納米粒子對人體健康和環(huán)境生物的潛在影響進行準確的評估。本文以斑馬魚(Daniorerio)和小球藻(Chlorella vulgaris)為生物模型,對納米CoFe_2O_4 的水生生物毒性進行研究,內(nèi)容包括納米CoFe_2O_4 引起的氧化應激、遺傳毒性、內(nèi)分泌干擾效應、納米CoFe_2O_4 的環(huán)境降解、納米中離子釋放以及生物自身對納米CoFe_2O_4 的抵抗機制等;此外,本文研究了納米CoFe_2O_4 與牛血清白蛋白(BSA)和酸性磷酸酶(AP)間的相互作用,探究了其熱力學數(shù)據(jù)及蛋白電暈的形成。將斑馬魚胚胎置于環(huán)境劑量的納米CoFe_2O_4 培養(yǎng)液中,分別暴露96和168hpf(hours post fertilization),暴露96和168 hpf,會引起嚴重的心包水腫、代謝降低、孵化延遲、尾部/脊柱彎曲以及細胞凋亡,且與CoFe_2O_4 呈劑量-效應與時間-效應關系。較低濃度的納米CoFe_2O_4 會引起過量ROS,進而引起頭部、心臟、尾部的細胞凋亡以及生物體內(nèi)DNA和代謝的變化。暴露168 hpf時,甲狀腺激素紊亂、納米粒子的團聚及粒子釋放會導致甲狀腺軸的膜損傷、氧化應激及結構損傷。且斑馬魚幼魚體內(nèi)T3、T4激素含量升高,導致孵化延遲、眼部、頭部畸形等現(xiàn)象。此外,ROS升高會引起8-OHd G DNA聚合物形成,引起DNA損傷,從而產(chǎn)生基因毒性。通過在藻細胞表面的吸附、聚集、釋放Fe3+和Co2+以及造成機械損傷,納米CoFe_2O_4 會損害小球藻細胞形態(tài)、膜完整性和通透性。結果也表明納米CoFe_2O_4 引起的ROS會引起細胞內(nèi)的氧化應激,導致CAT、GST、AP等抗氧化酶活性下降,并引起遺傳突變、代謝及細胞信號傳遞紊亂。納米CoFe_2O_4 濃度較低時對ROS含量、CAT、GST活性影響不顯著。這項研究表明,誘導ROS產(chǎn)生是CoFe_2O_4 NPs的致毒方式之一,并闡明了在自然環(huán)境中可能發(fā)生于生物體和納米顆粒之間的復雜過程。以光譜法作為手段,本文研究納米粒子與牛血清白蛋白(BSA)、酸性磷酸酶(AP)的相互作用,探討了納米CoFe_2O_4 對蛋白質(zhì)結構和功能的潛在影響作用。結果表明,納米CoFe_2O_4 通過靜態(tài)猝滅機制引起B(yǎng)SA和AP的熒光猝滅。負值熱力學參數(shù)(ΔH和ΔG)說明這種靜態(tài)猝滅是自發(fā)和放熱的。ΔS的負值和正值,表明CoFe_2O_4 NPs與BSA和AP間的結合力分別為范德華力、氫鍵和靜電作用。此外,通過TGA、DLS測試證明BSA和AP在CoFe_2O_4 NPs上形成了蛋白質(zhì)電暈,BSA和AP在CoFe_2O_4 NPs表面的密集包覆,使得負的zeta電位上升。BSA和AP在CoFe_2O_4 NPs上的這種包覆使得磁性飽和值從50.4 emu分別下降到了46.2和45.5 emu。通過對比靜態(tài)熒光猝滅和理論分析值,進一步分析BSA在CoFe_2O_4 NPs上形成的蛋白質(zhì)電暈。利用FTIR、UV-CD、紫外可見分光光譜和三維光譜等方法證實CoFe_2O_4 NPs與蛋白質(zhì)的結合會引起B(yǎng)SA和AP內(nèi)部微環(huán)境改變,引起二級結構和三級結構的改變。此外,同步熒光(SFS)表明CoFe_2O_4 NPs明顯改變了BSA和AP內(nèi)部色氨酸(Trp)殘基附近的微環(huán)境。通過測定BSA酯酶活性,說明CoFe_2O_4 NPs會引起B(yǎng)SA變性。本文還進一步研究了CoFe_2O_4 NPs對小球藻中AP活性的影響,實驗分別測定了CoFe_2O_4 NPs濃度為0和200μM時小球藻液的表觀米氏常數(shù)(Km)和活化能,表觀米氏常數(shù)常數(shù)分別為0.57和26.5 m M,活化能分別為0.538和3.428 KJ mol-1。-7 Umml-1的表觀Vmax值說明酶活性位點完全被NPs占據(jù)而沒有給酶底物留下空間。結果表明CoFe_2O_4 NPs通過使AP酶展開而降低了酶活性,說明CoFe_2O_4 NPs會通過改變AP酶結構和代謝活性而破壞酶的活性。本文從多個層面闡述了納米CoFe_2O_4 的生物毒性,為開發(fā)環(huán)境友好、安全的納米材料提供了理論性依據(jù)。同時,提供了能更好地精確控制和分析納米顆粒在復雜的生物和環(huán)境體系中的方法,為相關監(jiān)管機構和部門制定和實施嚴格的法規(guī)提供了參考。
[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.

【學位授予單位】：浙江工業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：TQ138.12;TB383.1
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本文編號：1835093