纳米氧化锌对HEK293细胞的毒性作用及其作用机制的研究

发布时间：2018-04-28 18:30

本文选题：纳米氧化锌 + HEK293细胞　；参考：《苏州大学》2015年硕士论文

【摘要】：纳米氧化锌(Zn O Nanoparticles,Zn O NPs)是目前继碳纳米管之后又一个备受关注的多功能纳米材料。纳米氧化锌的颗粒大小约在1~100nm之间。由于晶粒的细微化,其表面电子结构和晶体结构发生变化,产生了宏观物体所不具有的表面效应、体积效应、量子尺寸效应和宏观隧道效应以及高透明度、高分散性等特点,而被广泛应用于橡胶、陶瓷、化工、环保、饲料、电子、化妆品、光学、生物、医药等众多领域,具有非常广阔的应用前景。纳米氧化锌在创造巨大经济效益的同时,其毒性和健康效应也应引起人们的关注。但是,到目前为止,关于纳米氧化锌的生物安全性的研究还很少,而且已取得的研究结果也并不统一,还需要进行大量的研究证明。基于此,本研究以三种不同尺寸的纳米氧化锌(15nm、30nm、90nm)为对象,比较不同浓度的纳米氧化锌(15nm)对HEK293细胞的毒性大小以及同一浓度(4.0μg/ml)不同尺寸的氧化锌(常规氧化锌、15nm、30nm、90nm)对HEK293细胞的毒性差异,并进一步研究纳米氧化锌的毒性作用机制,以期为纳米氧化锌做体外安全性评估,并为纳米材料毒理学以及其生物安全性研究提供一定的理论依据。首先,利用透射电子显微镜(TEM)和动态光散射(DLS)对实验所用纳米氧化锌(15nm、30nm、90nm)进行表征,然后分别从纳米氧化锌对HEK293细胞的细胞形态学、细胞损伤、抗氧化指标等方面的影响进行研究。研究结果显示,实验所用3种不同尺寸Zn O NPs的纯度均大于99.9%,其结构均为红锌矿晶型,其尺寸均比理论尺寸略大。用15nm不同浓度(0、0.5、1.0、2.0、4.0、8.0、16.0μg/ml)的Zn O NPs、同一浓度(4.0μg/ml)不同尺寸(常规Zn O、15nm、30nm、90nm)的Zn O NPs处理HEK293细胞,培养48h后,荧光显微镜观察显示,随着Zn O NPs浓度的逐渐增大,HEK293细胞逐渐出现形态学变化:细胞排列疏松,间隙增大,胞质内颗粒物增多,透明度下降,部分细胞回缩变圆并出现脱落。且在相同浓度下,Zn O NPs尺寸越小,这种变化越明显。MTT、流式细胞仪、Caspase3检测结果显示,随着Zn O NPs浓度的增大,HEK293细胞活性逐渐降低,凋亡率逐渐升高,Caspase3活性逐渐增强;随着Zn O NPs尺寸的减小,HEK293细胞活性逐渐降低,凋亡率逐渐升高,Caspase3活性逐渐增强。HEK293细胞抗氧化指标(MDA、ROS、GSH)检测结果显示,随着Zn O NPs浓度的增大,HEK293细胞中MDA和ROS水平逐渐升高,GSH水平逐渐降低;随着Zn O NPs尺寸的减小,HEK293细胞中MDA和ROS水平逐渐升高,GSH水平逐渐降低。MDA、ROS的过量积累,GSH的大量消耗,影响了细胞正常的氧化-抗氧化水平,诱导HEK293细胞出现氧化应激状态,从而进一步诱导Caspase3的活化,最后导致细胞发生凋亡,且Zn O NPs对HEK293细胞的这种毒性作用具有明显的剂量效应和尺寸效应,即Zn O NPs浓度越大,其对HEK293细胞的毒性越大,Zn O NPs尺寸越小,其对HEK293细胞的毒性越大。
[Abstract]:Nano-ZnO Zn-O Nanoparticles-Zn-O NPsis another multifunctional nano-material, which has attracted much attention after carbon nanotubes (CNTs). The particle size of nanometer zinc oxide is about between 1~100nm. Due to the fineness of the grain, the surface electronic structure and crystal structure change, resulting in the surface effect, volume effect, quantum size effect, macroscopic tunnel effect, high transparency, high dispersion and so on. It is widely used in many fields, such as rubber, ceramics, chemical industry, environmental protection, feed, electronics, cosmetics, optics, biology, medicine and so on. At the same time, the toxicity and health effect of nanometer zinc oxide should be paid more attention to. However, so far, there are few studies on the biosafety of nano-zinc oxide, and the results obtained are not uniform, and a lot of research is needed to prove it. Based on this, three kinds of nanometer zinc oxide with different sizes were studied. To compare the toxicity of different concentrations of nanometer zinc oxide (15 nm) to HEK293 cells and the toxicity of different sizes of zinc oxide (conventional zinc oxide 15 nm / ml) to HEK293 cells, and to further study the mechanism of the toxicity of nanometer zinc oxide. In order to do in vitro safety evaluation of nano-zinc oxide, and provide a theoretical basis for the toxicology of nano-materials and their biological safety research. First, transmission electron microscopy (TEM) and dynamic light scattering (DLSs) were used to characterize the HEK293 cells. The effects of antioxidant indexes were studied. The results show that the purity of three different sizes of Zno NPs is greater than 99.9, and the structure of all of them is red zinc ore crystal type, which is slightly larger than the theoretical size. HEK293 cells were treated with Zn O NPs with different concentrations of Zn O NPs, of different concentration (15nm) of 0.5nmnmnmnmnmnmnmnm ~ (30 nm) and 4.0 渭 g / ml of 4.0 渭 g / ml of Zn O NPs, (4.0 渭 g / ml). The cells were cultured for 48 h, and the results of fluorescence microscopy showed that the HEK293 cells were cultured for 48 hours, and the results were as follows: (1) after 48 h culture, the cells were treated with Zn O NPs of the same concentration of 4.0 渭 g / ml. With the increasing of Zn O NPs concentration, morphological changes of HEK293 cells appeared gradually: the arrangement of cells was loose, the gap was enlarged, the particles in the cytoplasm were increased, the transparency was decreased, and some cells were reduced to round and shedding. The smaller the size of Zn-O NPs at the same concentration, the more obvious the change was. The results of flow cytometry showed that the activity of HEK293 cells decreased with the increase of Zn O NPs concentration, and the apoptosis rate increased gradually. With the decrease of Zno NPs size, the activity of HEK293 cells decreased, the apoptosis rate increased and the activity of Caspase3 increased. The results showed that the antioxidant index of HEK293 cells was determined by MDA-ROSS-GSH. With the increase of Zn O NPs concentration, the levels of MDA and ROS in HEK293 cells gradually increased, and the levels of MDA and ROS in HEK293 cells gradually decreased with the decrease of Zn O NPs size. It affected the normal level of oxidation-antioxidation, induced oxidative stress in HEK293 cells, further induced the activation of Caspase3, and finally resulted in cell apoptosis. The toxicity of Zn O NPs to HEK293 cells showed significant dose and size effects, that is, the greater the concentration of Zn O NPs, the greater the size of Zn O NPs and the greater the toxicity to HEK293 cells.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1;X171.5

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