体外研究羧基化多壁碳纳米管诱发的氧化应激效应与机理

发布时间：2018-10-16 11:35

【摘要】：自从1991年碳纳米管发现以来,由于其具有奇特的电学性质、光学和光电子性质、力学和机电性质、磁学和电磁性质以及热学和热电性质,因而在诸多领域都显现出广阔的应用潜力。与此同时,碳纳米管的毒性作用也备受关注。碳纳米管通常分为单壁碳纳米管和多壁碳纳米管。由于多壁碳纳米管具有较强的生物相容性,进而,多壁碳纳米管(MWCNTs)的安全性受到了更多的关注。因此,本论文选用分散性较好的羧基化多壁碳纳米管(MWCNTs-COOH)展开研究。有研究指出,纳米材料进入生物体产生的毒性作用,与诱发氧化应激效应相关。氧化应激效应将会导致机体内活性氧(ROS)的大量产生。活性氧(ROS)在体内积聚达到一定的量,机体将产生氧化应激损伤,进而显现出各类病症。因此,通过研究碳纳米管诱发机体产生的氧化应激效应与机理,将有助于进一步深入研究和认识碳纳米管生物毒性的作用机理,为今后对碳纳米管毒性作用的研究提供更多的科学依据。本论文将结合分子水平和细胞水平,从体外研究羧基化多壁碳纳米管诱发的氧化应激效应与机理。研究的主要内容如下：首先,通过红外光谱和高分辨透射电镜对MWCNTs-COOH进行了表征。其次,通过多种光谱学的方法和等温滴定量热法等手段,研究了MWCNTs-COOH结果表明,MWCNTs-COOH与两种酶的相互作用都比较弱,且都是以疏水作用力为主的弱吸热反应。MWCNTs-COOH对两种酶的结构均没有产生明显的影响。酶活性实验表明,MWCNTs-COOH未对它们的生理功能产生明显的影响。最后,利用Muse智能触控细胞分析仪,研究了分子水平浓度下MWCNTs-COOH对原代小鼠肝细胞成活率的影响。以此为基础,测定了MWCNTs-COOH对细胞内过氧化酶、超氧化物歧化酶活性和丙二醛(MDA)含量的影响,探讨了MWCNTs-COOH诱发细胞产生的氧化应激效应。主要结论：①WCNTs-COOH对细胞活力呈时间依赖效应；②CAI、SOD的活性都受到了抑制,MWCNTs-COOH诱发肝细胞产生氧化应激；③MDA的含量发生变化,但没有显著性差异,说明MWCNTs-COOH未对细胞造成明显的氧化应激损伤。通过对比两种不同研究手段的实验结果发现：在相同的MWCNTs-COOH浓度下,分子和细胞水平上抗氧化应激关键酶活性的变化不一致,揭示出MWCNTs-COOH不是直接作用于抗氧化应激关键酶,通过影响其结构而改变其生理活性；而是通过诱发氧化应激效应,间接影响抗氧化应激关键酶的生理活性。细胞内抗氧化应激关键酶的活性与氧化应激效应有着复杂的关联,这将为今后深入研究碳纳米管的生物毒性作用提供更多的参考依据。
[Abstract]:Since the discovery of carbon nanotubes in 1991, due to their unique electrical, optical and optoelectronic properties, mechanical and mechanical properties, magnetic and electromagnetic properties, and thermal and thermoelectric properties, As a result, there are broad application potential in many fields. At the same time, the toxicity of carbon nanotubes (CNTs) has attracted much attention. Carbon nanotubes are usually divided into single-walled carbon nanotubes and multi-walled carbon nanotubes. The safety of multiwalled carbon nanotubes (MwCNTs) has attracted more and more attention due to their strong biocompatibility. Therefore, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) with good dispersity were selected in this thesis. Some studies have indicated that the toxic effect of nanomaterials entering organism is related to the effect of induced oxidative stress. Oxidative stress will lead to the production of reactive oxygen species (ROS) in the body. Reactive oxygen species (Ros) (ROS) accumulates to a certain amount in the body, which will lead to oxidative stress damage, and then show various diseases. Therefore, by studying the oxidative stress effect and mechanism induced by carbon nanotubes, it will be helpful to further study and understand the mechanism of biological toxicity of carbon nanotubes. It provides more scientific basis for the study of the toxicity of carbon nanotubes in the future. In this paper, the oxidative stress induced by carboxylated multiwalled carbon nanotubes (MMWNTs) was studied in vitro at the molecular and cellular levels. The main contents are as follows: firstly, MWCNTs-COOH was characterized by infrared spectroscopy and high resolution transmission electron microscope. Secondly, by means of various spectroscopic methods and isothermal drop quantitative calorimetry, the MWCNTs-COOH results show that the interaction between MWCNTs-COOH and the two enzymes is weak. Both of them were weak endothermic reactions dominated by hydrophobic forces. MWCNTs-COOH had no obvious effect on the structure of the two enzymes. The enzyme activity experiment showed that MWCNTs-COOH had no obvious effect on their physiological function. Finally, the effect of MWCNTs-COOH on the survival rate of primary mouse hepatocytes at molecular level was studied by Muse intelligent touch cell analyzer. On this basis, the effects of MWCNTs-COOH on the activities of peroxidase, superoxide dismutase and malondialdehyde (MDA) in cells were determined, and the oxidative stress induced by MWCNTs-COOH was discussed. Conclusion: 1WCNTs-COOH has a time-dependent effect on cell viability, the activity of 1WCNTs-COOH is inhibited, MWCNTs-COOH induces oxidative stress in hepatocytes, the content of 3MDA changes, but there is no significant difference. The results showed that MWCNTs-COOH did not cause obvious oxidative stress damage to cells. By comparing the results of two different research methods, it was found that at the same concentration of MWCNTs-COOH, the changes of the key enzymes of antioxidant stress at the molecular and cellular levels were not consistent, which revealed that MWCNTs-COOH did not act directly on the key enzymes of antioxidant stress. The physiological activity was changed by affecting its structure, and the physiological activity of key enzymes of antioxidant stress was indirectly affected by inducing oxidative stress effect. The activities of the key enzymes of intracellular antioxidant stress are closely related to the oxidative stress effect, which will provide more references for the further study of the biological toxicity of carbon nanotubes in the future.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：O613.71;TB383.1

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