本文選題：金屬納米氧化物 + 維生素C��； 參考：《上海大學》2013年碩士論文

【摘要】：隨著納米技術(shù)的飛速發(fā)展，金屬納米氧化物已經(jīng)在食品添加劑領(lǐng)域得到了廣泛的應(yīng)用，并引起了科學界的關(guān)注。鋅、硅、銅、鈦等的金屬納米氧化物在食品安全技術(shù)領(lǐng)域在一定用量范圍內(nèi)是允許被使用的。食品和藥品管理局（FDA）已經(jīng)將氧化鋅（ZnO）列為食品添加劑中普遍公認的安全材料。自從1990年以來，二氧化鈦（TiO2）作為白色顏料廣泛應(yīng)用于食品添加劑領(lǐng)域，例如，奶酪、糖果、白巧克力。目前，食品添加劑安全評估標準往往是基于單獨添加劑的毒性效應(yīng)建立起來的，而人們每天攝入的食物種類繁多，一種食品中往往存在多種食品添加劑聯(lián)合使用的情況。因此，正確評價食品添加劑的聯(lián)合毒性作用，對食品添加劑的安全使用具有重要意義。維生素C（VC）是生物液體合成和免疫系統(tǒng)功能中強有力的水溶性抗氧化劑，許多食品中都添加VC以提高人體免疫力。探究食品添加劑中金屬納米氧化物與VC對于胃腸道系統(tǒng)和神經(jīng)系統(tǒng)的聯(lián)合毒性是非常有必要的。由此，本研究使用人胃黏膜上皮細胞（GES-1）和神經(jīng)干細胞（C17.2）分別作為胃腸道系統(tǒng)和神經(jīng)系統(tǒng)的模型，采用檢測細胞成活率、細胞凋亡和死亡、細胞內(nèi)鈣離子濃度的變化以及觀察細胞形態(tài)等方法探究復(fù)雜體系的聯(lián)合毒性。并研究了不同種類、不同作用濃度、不同粒徑、不同性質(zhì)（親水或親油）的金屬納米氧化物的細胞毒理學影響。 首先，本論文利用透射電子顯微鏡、X射線熒光分析、X射線衍射技術(shù)、NTA、Zeta電位、紅外光譜等方法對所選樣品的粒徑、晶體結(jié)構(gòu)、純度、培養(yǎng)基和水中樣品的粒徑及表面電勢、樣品表面性質(zhì)等進行了表征。本研究應(yīng)用了六種氧化鋅納米顆粒（ZnO-NPs），分別命名為ZnO-1（納米顆粒平均直徑為20納米，親水性）、ZnO-2（納米顆粒平均直徑為20納米，親油性）、ZnO-3（納米顆粒平均直徑為100納米，親水性）、ZnO-4（納米顆粒平均直徑為100納米，親油性）、ZnO-5（納米顆粒平均直徑為100納米，食品級）和ZnO-6（納米顆粒的平均粒徑為100納米，食品級），且ZnO-5、ZnO-6來自不同的制造商。應(yīng)用了四種二氧化鈦納米顆粒（TiO2-NPs），分別命名為TiO2-1（納米顆粒平均直徑20納米，親水性）， TiO2-2（納米顆粒平均直徑20納米，親油性），TiO2-3（納米顆粒平均直徑50納米，高分散食品級）和TiO2-4（納米顆粒的平均粒徑為50納米，食品級）。TEM照片顯示了ZnO-NPs和TiO2-NPs的形貌和粒徑是不同的，而NTA實驗結(jié)果顯示，相同樣品在水中或培養(yǎng)基中的粒徑是相似的。據(jù)X射線熒光（光譜儀）分析結(jié)果，所有納米材料的純度均高于95%�；赬射線衍射（XRD）檢測，六種氧化鋅皆六方晶型，二氧化鈦為銳鈦礦型。 在此基礎(chǔ)上，本文研究了金屬納米氧化物與VC復(fù)合體系對細胞的聯(lián)合毒性作用。研究表明，單獨ZnO-NPs作用濃度小于15μg/mL時，對兩種細胞均沒有顯著的毒性效應(yīng)，且單獨VC作用濃度小于300μg/mL時對細胞也是安全的。當ZnO-NPs的濃度為15μg/mL時，GES-1細胞和C17.2細胞的存活率均高于90%，當VC濃度為300μg/mL時，GES-1細胞和C17.2細胞的存活率均高于95%，然而，當15μg/mL的ZnO-NPs與300μg/mL的VC共同作用時，VC能夠加強氧化鋅對于細胞（GES-1和C17.2）的毒性，細胞的成活率在30%左右。通過凋亡對比實驗可以發(fā)現(xiàn)，細胞染毒24h后，單獨的VC (300μg/mL)和單獨的ZnO-5(15μg/mL)均沒有引起細胞（GES-1和C17.2）早期凋亡而產(chǎn)生明顯變化，但是ZnO-5(15μg/mL)VC (300μg/mL)組的凋亡率與對照組相比顯著上升，對GES-1細胞的凋亡率高達52.17%，死亡率達5.46%；對C17.2細胞的凋亡率高達51.38%，死亡率達9.44%。使用Fluo-3探針檢測暴露于單獨的ZnO-5(15μg/mL)，單獨的VC (300μg/mL)以及ZnO-5(15μg/mL)VC (300μg/mL)2h后的三組GES-1和C17.2細胞，與對照組相比，單獨的ZnO-5(15μg/mL)和VC(300μg/mL)均沒有引起細胞內(nèi)鈣離子的明顯變化，但是ZnO-5(15μg/mL)VC(300μg/mL)組細胞內(nèi)鈣離子濃度顯著上升。通過激光掃描共聚焦顯微鏡Confocal拍攝處理后的GES-1和C17.2細胞，結(jié)果顯示與空白組對比，，單獨的ZnO-5(15μg/mL)和單獨的VC (300μg/mL)均沒有引起細胞核的顯著性變形或死亡，但是ZnO-NPs（15μg/mL）VC (300μg/mL)組的細胞核卻顯著變形，說明大部分細胞已經(jīng)死亡。但同樣作為金屬納米氧化物的二氧化鈦與VC共同作用時，VC對細胞沒有表現(xiàn)出聯(lián)合毒性效應(yīng)。 最后，本文探究了兩種金屬納米氧化物產(chǎn)生不同結(jié)論的原因。研究發(fā)現(xiàn)，由于ZnO-NPs極易與VC發(fā)生反應(yīng)，使得30μg/mL的氧化鋅溶液中Zn~(2+)的溶解度由17μg/mL增加到29μg/mL，由于氧化鋅對細胞的毒性主要是由Zn~(2+)引起的，該變化使其對細胞的影響加劇。另一方面，VC與ZnO-NPs反應(yīng)致使ZnO-NPs的團聚減小，從而更容易進入細胞，對細胞造成一定程度的損傷。 本文為今后復(fù)合體系中金屬納米材料毒性和食品安全方面的研究提供了研究方法和重要數(shù)據(jù)基礎(chǔ)，對復(fù)雜體系領(lǐng)域的研究具有重要意義。
[Abstract]:With the rapid development of nanotechnology, the metal oxide has been widely used in the field of food additives and attracted the attention of the scientific community. The metal oxides of zinc, silicon, copper and titanium are allowed to be used in the field of food safety technology. The food and Drug Administration (FDA) has already made oxygen. Zinc (ZnO) is generally recognized as a safety material in food additives. Since 1990, titanium dioxide (TiO2) has been widely used as a white pigment in the field of food additives, such as cheese, candy, and white chocolate. At present, the safety assessment standard of food additives is established on the basis of the toxic effects of individual additives. There are a wide variety of food intake every day, and a variety of food additives often exist in one kind of food. Therefore, it is of great significance to correctly evaluate the combined toxicity of food additives for the safe use of food additives. Vitamin C (VC) is a strong water-soluble resistance in the function of biological liquid synthesis and immune system. VC is added in many foods to improve human immunity. It is necessary to explore the joint toxicity of metal nanometers and VC in the gastrointestinal system and the nervous system in food additives. Thus, this study uses human gastric mucosal epithelial cells (GES-1) and neural stem cells (C17.2) as gastrointestinal systems and nerves, respectively. A systematic model to investigate the joint toxicity of complex systems by detecting cell survival rate, apoptosis and death, changes in intracellular calcium concentration and observing cell morphology, and studying the Cellular Toxicology of different types, different concentrations, different particle sizes, and different properties (water pro or oil). Ringing.
Firstly, this paper uses transmission electron microscopy, X ray fluorescence analysis, X ray diffraction, NTA, Zeta potential, infrared spectroscopy to characterize the particle size, crystal structure, purity, medium, surface potential and surface properties of samples in water, and the surface properties of samples. This study applied six kinds of Zinc Oxide nanoparticles (Zn O-NPs), respectively named ZnO-1 (the average diameter of nanoparticles are 20 nanometers, hydrophilic), ZnO-2 (the average diameter of nanoparticles are 20 nanometers, pro oiliness), ZnO-3 (the average diameter of nanoparticles is 100 nanometers, hydrophilic), ZnO-4 (the average diameter of nanoparticles is 100 nanometers, pro oiliness), ZnO-5 (the average diameter of nanoparticles is 100 nanometers, food grade) and ZnO-6 (the average diameter of nanoparticles is 100 nanometers, food grade) and ZnO-6. The average particle size of the nanoparticles was 100 nanometers, food grade, and ZnO-5, and ZnO-6 came from different manufacturers. Four kinds of titanium dioxide nanoparticles (TiO2-NPs) were used, named TiO2-1 (the average diameter of nanoparticles 20 nanometers, hydrophilic), TiO2-2 (nanometers flat average diameter 20 nanometers, oiliness), and TiO2-3 (average diameter of nanoparticles 50 nanometers, High dispersion food grade and TiO2-4 (the average particle size of 50 nanoparticles, food grade).TEM photographs show that the morphology and particle size of ZnO-NPs and TiO2-NPs are different, and the NTA test results show that the size of the same sample in water or medium is similar. According to the results of the X ray fluorescence (spectrometer) analysis, the purity of all nanomaterials is all Above 95%., based on X ray diffraction (XRD), six types of Zinc Oxide have six square crystals and titanium dioxide is anatase.
On this basis, this paper studies the joint toxic effects of the metal nano oxide and the VC composite system on the cells. The study shows that when the concentration of ZnO-NPs is less than 15 u g/mL, there is no significant toxic effect on the two cells, and the cells are also safe when the concentration of VC is less than 300 u g/mL. When the concentration of ZnO-NPs is 15 u g/mL The survival rate of GES-1 cells and C17.2 cells were higher than 90%. When VC concentration was 300 g/mL, the survival rate of GES-1 cells and C17.2 cells was higher than 95%. However, when ZnO-NPs of 15 mu g/mL combined with 300 micron VC, VC could enhance the toxicity of Zinc Oxide to cells (GES-1 and cells), and the survival rate of cells was about 30%. Compared with the experiment, it was found that after the cells were infected with 24h, VC (300 mu g/mL) and individual ZnO-5 (15 mu g/mL) did not cause significant changes in the early apoptosis of cells (GES-1 and C17.2), but the apoptosis rate of ZnO-5 (15) VC (300) VC (300 Mu g/mL) group was significantly higher than that of the control group, and the apoptosis rate of the GES-1 cells was up to 52.17% and the mortality rate was 5.46%. The apoptosis rate of C17.2 cells was up to 51.38%, and the mortality rate was 9.44%. using Fluo-3 probe to detect ZnO-5 (15 g/mL), VC (300 mu g/mL) and ZnO-5 (15 g/mL) VC (300 mu g/mL) 2H cells. Compared with the control group, the individual (15 mu) and 300 mu (300 mu) did not cause intracellular calcium ionization. The intracellular calcium concentration in the ZnO-5 (15 mu g/mL) VC (300 g/mL) group increased significantly. The GES-1 and C17.2 cells were taken after the laser scanning confocal microscopy (confocal microscopy) Confocal. The results showed that the single ZnO-5 (15 u g/mL) and single VC (300 mu g/mL) were not caused by the significant deformation of the nucleus. Or death, but the nuclei of the ZnO-NPs (15 u g/mL) VC (300 g/mL) group were significantly deformed, indicating that most of the cells were dead. But as the same as the metal oxide titanium dioxide and VC, VC did not exhibit a joint toxic effect on the cells.
Finally, this paper explores the reasons for the different conclusions of the two metal nanometers. It was found that the solubility of Zn~ (2+) in the 30 g/mL Zinc Oxide solution increased from 17 to 29 g/mL because of its susceptibility to VC, because the toxicity of Zinc Oxide to the cells was mainly caused by Zn~ (2+), which made it to the cell. On the other hand, the reaction of VC and ZnO-NPs reduced the aggregation of ZnO-NPs, which made it easier to enter cells and cause some damage to cells.
This paper provides a research method and important data base for the research of the toxicity and food safety of metal nanomaterials in the composite system in the future. It is of great significance to the research in the field of complex systems.
【學位授予單位】：上海大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

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