纳米金对P450酶抑制机理的研究

发布时间：2018-10-12 16:38

【摘要】：随着纳米技术的发展,金纳米粒子因其特殊性能已成功应用于生物医药领域,如艾滋病、肿瘤和帕金森氏症等疾病的治疗；另外,越来越多的含金纳米粒子的商品出现在货架上,使得人体通过皮肤、肺、胃肠道接触到它并且造成健康伤害的机会增高。作为一种特殊的外源物质,金纳米粒子一旦进入机体,就会与细胞色素P450酶(CYPs)接触。CYPs是一个含有多种亚型的超家族酶系,主要在肝脏表达,在大多数环境毒物、药物代谢和解毒中扮演着重要角色。因此,深入研究纳米金与CYPs相互作用及其生物暴露对CYPs的功能影响有重要意义,目前此方面的研究较少。本论文探讨了纳米金粒子对人肝微粒体中主要CYP同工酶介导的Ⅰ相药物代谢的影响及其机理,开展了以下工作： (1)为了评价CYP同工酶的活性变化,建立了一个能同时定量六种代谢物的LC-MS/MS分析方法,并考察了此方法的准确性与精密度。同时,采用了一种简便且高效的生物样品前处理方法,即盐诱导分离纳米粒子,经此方法处理后,生物样品可直接进入LC-MS/MS分析。 (2)以六种药物为探针底物,在体外孵育体系中,系统考察了不同尺寸的金纳米粒子(5～100nm)对5种CYP同工酶的生物转化活性的影响,发现纳米金对2C9、2C19、2D6、3A4有明显抑制作用,且抑制作用呈尺寸和浓度依赖的特点,即随纳米金粒径减小或浓度增大而增强。 (3)为探讨抑制机理,在微粒体孵育动态过程中,采用紫外-可见光谱、动态光散射、zeta电位表征了纳米金的表面性质及其改变,发现孵育过程中,纳米金的等离子共振吸收峰发生了红移、粒径增大、表面电荷降低,表明酶蛋白与纳米粒子发生结合。进一步测得能使7或70nm纳米金粒径增大最多、红移最多的人肝微粒体的临界浓度。由此推测人肝微粒体的膜结构能在纳米金表面形成微囊,从而影响了膜的完整性,导致固定在膜上面的CYPs酶活性的改变。
[Abstract]:With the development of nanotechnology, gold nanoparticles have been successfully used in biomedical fields such as AIDS, cancer and Parkinson's disease because of their special properties. More and more gold nanoparticles appear on shelves, increasing the chances that the body will touch it through the skin, lungs, and gastrointestinal tract and cause health damage. As a special exogenous substance, gold nanoparticles come into contact with cytochrome P450 enzyme (CYPs) once they enter the body. CYPs is a superfamily of enzymes containing many subtypes, mainly expressed in the liver and most of the environmental poisons. Drug metabolism and detoxification play an important role. Therefore, it is of great significance to study the interaction of gold nanoparticles with CYPs and the effects of biological exposure on the function of CYPs. In this paper, the effects of gold nanoparticles on the metabolism of phase I drugs mediated by the main CYP isozymes in human liver microsomes were investigated. The following works were carried out: (1) to evaluate the activity of CYP isozymes; A LC-MS/MS method for simultaneous quantification of six metabolites was established and its accuracy and precision were investigated. At the same time, a simple and efficient pretreatment method for biological samples was adopted, that is, salt induced separation of nanoparticles. After this method, biological samples could be directly analyzed by LC-MS/MS. (2) six drugs were used as probe substrates. In vitro incubation system, the effects of different sizes of gold nanoparticles (5~100nm) on the biotransformation activities of five CYP isozymes were systematically investigated. (3) in order to investigate the inhibition mechanism, UV-Vis spectroscopy, dynamic light scattering and zeta potential were used to characterize the surface properties and changes of nanocrystalline gold during microsomal incubation. It is found that the plasmon resonance absorption peak of gold nanoparticles is red-shifted, the particle size increases and the surface charge decreases during incubation, which indicates that the enzyme protein binds to the nanoparticles. Furthermore, the critical concentration of human liver microsomes with 7 or 70nm nanocrystalline gold particle size increased most and redshift most. It is inferred that the membrane structure of human liver microsomes can form microcapsules on the surface of gold nanoparticles, which affects the integrity of the membrane and results in the change of CYPs enzyme activity immobilized on the membrane.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TB383.1;R96

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