食品添加剂中的金属纳米氧化物与维生素C对于胃肠道系统和神经系统的复合毒性效应研究

发布时间：2018-07-05 01:05

本文选题：金属纳米氧化物 + 维生素C　；参考：《上海大学》2013年硕士论文

【摘要】：随着纳米技术的飞速发展，金属纳米氧化物已经在食品添加剂领域得到了广泛的应用，并引起了科学界的关注。锌、硅、铜、钛等的金属纳米氧化物在食品安全技术领域在一定用量范围内是允许被使用的。食品和药品管理局（FDA）已经将氧化锌（ZnO）列为食品添加剂中普遍公认的安全材料。自从1990年以来，二氧化钛（TiO2）作为白色颜料广泛应用于食品添加剂领域，例如，奶酪、糖果、白巧克力。目前，食品添加剂安全评估标准往往是基于单独添加剂的毒性效应建立起来的，而人们每天摄入的食物种类繁多，一种食品中往往存在多种食品添加剂联合使用的情况。因此，正确评价食品添加剂的联合毒性作用，对食品添加剂的安全使用具有重要意义。维生素C（VC）是生物液体合成和免疫系统功能中强有力的水溶性抗氧化剂，许多食品中都添加VC以提高人体免疫力。探究食品添加剂中金属纳米氧化物与VC对于胃肠道系统和神经系统的联合毒性是非常有必要的。由此，本研究使用人胃黏膜上皮细胞（GES-1）和神经干细胞（C17.2）分别作为胃肠道系统和神经系统的模型，采用检测细胞成活率、细胞凋亡和死亡、细胞内钙离子浓度的变化以及观察细胞形态等方法探究复杂体系的联合毒性。并研究了不同种类、不同作用浓度、不同粒径、不同性质（亲水或亲油）的金属纳米氧化物的细胞毒理学影响。首先，本论文利用透射电子显微镜、X射线荧光分析、X射线衍射技术、NTA、Zeta电位、红外光谱等方法对所选样品的粒径、晶体结构、纯度、培养基和水中样品的粒径及表面电势、样品表面性质等进行了表征。本研究应用了六种氧化锌纳米颗粒（ZnO-NPs），分别命名为ZnO-1（纳米颗粒平均直径为20纳米，亲水性）、ZnO-2（纳米颗粒平均直径为20纳米，亲油性）、ZnO-3（纳米颗粒平均直径为100纳米，亲水性）、ZnO-4（纳米颗粒平均直径为100纳米，亲油性）、ZnO-5（纳米颗粒平均直径为100纳米，食品级）和ZnO-6（纳米颗粒的平均粒径为100纳米，食品级），且ZnO-5、ZnO-6来自不同的制造商。应用了四种二氧化钛纳米颗粒（TiO2-NPs），分别命名为TiO2-1（纳米颗粒平均直径20纳米，亲水性）， TiO2-2（纳米颗粒平均直径20纳米，亲油性），TiO2-3（纳米颗粒平均直径50纳米，高分散食品级）和TiO2-4（纳米颗粒的平均粒径为50纳米，食品级）。TEM照片显示了ZnO-NPs和TiO2-NPs的形貌和粒径是不同的，而NTA实验结果显示，相同样品在水中或培养基中的粒径是相似的。据X射线荧光（光谱仪）分析结果，所有纳米材料的纯度均高于95%。基于X射线衍射（XRD）检测，六种氧化锌皆六方晶型，二氧化钛为锐钛矿型。在此基础上，本文研究了金属纳米氧化物与VC复合体系对细胞的联合毒性作用。研究表明，单独ZnO-NPs作用浓度小于15μg/mL时，对两种细胞均没有显著的毒性效应，且单独VC作用浓度小于300μg/mL时对细胞也是安全的。当ZnO-NPs的浓度为15μg/mL时，GES-1细胞和C17.2细胞的存活率均高于90%，当VC浓度为300μg/mL时，GES-1细胞和C17.2细胞的存活率均高于95%，然而，当15μg/mL的ZnO-NPs与300μg/mL的VC共同作用时，VC能够加强氧化锌对于细胞（GES-1和C17.2）的毒性，细胞的成活率在30%左右。通过凋亡对比实验可以发现，细胞染毒24h后，单独的VC (300μg/mL)和单独的ZnO-5(15μg/mL)均没有引起细胞（GES-1和C17.2）早期凋亡而产生明显变化，但是ZnO-5(15μg/mL)VC (300μg/mL)组的凋亡率与对照组相比显著上升，对GES-1细胞的凋亡率高达52.17%，死亡率达5.46%；对C17.2细胞的凋亡率高达51.38%，死亡率达9.44%。使用Fluo-3探针检测暴露于单独的ZnO-5(15μg/mL)，单独的VC (300μg/mL)以及ZnO-5(15μg/mL)VC (300μg/mL)2h后的三组GES-1和C17.2细胞，与对照组相比，单独的ZnO-5(15μg/mL)和VC(300μg/mL)均没有引起细胞内钙离子的明显变化，但是ZnO-5(15μg/mL)VC(300μg/mL)组细胞内钙离子浓度显著上升。通过激光扫描共聚焦显微镜Confocal拍摄处理后的GES-1和C17.2细胞，结果显示与空白组对比，，单独的ZnO-5(15μg/mL)和单独的VC (300μg/mL)均没有引起细胞核的显著性变形或死亡，但是ZnO-NPs（15μg/mL）VC (300μg/mL)组的细胞核却显著变形，说明大部分细胞已经死亡。但同样作为金属纳米氧化物的二氧化钛与VC共同作用时，VC对细胞没有表现出联合毒性效应。最后，本文探究了两种金属纳米氧化物产生不同结论的原因。研究发现，由于ZnO-NPs极易与VC发生反应，使得30μg/mL的氧化锌溶液中Zn~(2+)的溶解度由17μg/mL增加到29μg/mL，由于氧化锌对细胞的毒性主要是由Zn~(2+)引起的，该变化使其对细胞的影响加剧。另一方面，VC与ZnO-NPs反应致使ZnO-NPs的团聚减小，从而更容易进入细胞，对细胞造成一定程度的损伤。本文为今后复合体系中金属纳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数据基础，对复杂体系领域的研究具有重要意义。
[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.
【学位授予单位】：上海大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R114

【参考文献】