【摘要】：隨著納米技術(shù)的發(fā)展,金納米粒子因其特殊性能已成功應(yīng)用于生物醫(yī)藥領(lǐng)域,如艾滋病、腫瘤和帕金森氏癥等疾病的治療；另外,越來越多的含金納米粒子的商品出現(xiàn)在貨架上,使得人體通過皮膚、肺、胃腸道接觸到它并且造成健康傷害的機會增高。作為一種特殊的外源物質(zhì),金納米粒子一旦進入機體,就會與細胞色素P450酶(CYPs)接觸。CYPs是一個含有多種亞型的超家族酶系,主要在肝臟表達,在大多數(shù)環(huán)境毒物、藥物代謝和解毒中扮演著重要角色。因此,深入研究納米金與CYPs相互作用及其生物暴露對CYPs的功能影響有重要意義,目前此方面的研究較少。本論文探討了納米金粒子對人肝微粒體中主要CYP同工酶介導(dǎo)的Ⅰ相藥物代謝的影響及其機理,開展了以下工作： (1)為了評價CYP同工酶的活性變化,建立了一個能同時定量六種代謝物的LC-MS/MS分析方法,并考察了此方法的準確性與精密度。同時,采用了一種簡便且高效的生物樣品前處理方法,即鹽誘導(dǎo)分離納米粒子,經(jīng)此方法處理后,生物樣品可直接進入LC-MS/MS分析。 (2)以六種藥物為探針底物,在體外孵育體系中,系統(tǒng)考察了不同尺寸的金納米粒子(5～100nm)對5種CYP同工酶的生物轉(zhuǎn)化活性的影響,發(fā)現(xiàn)納米金對2C9、2C19、2D6、3A4有明顯抑制作用,且抑制作用呈尺寸和濃度依賴的特點,即隨納米金粒徑減小或濃度增大而增強。 (3)為探討抑制機理,在微粒體孵育動態(tài)過程中,采用紫外-可見光譜、動態(tài)光散射、zeta電位表征了納米金的表面性質(zhì)及其改變,發(fā)現(xiàn)孵育過程中,納米金的等離子共振吸收峰發(fā)生了紅移、粒徑增大、表面電荷降低,表明酶蛋白與納米粒子發(fā)生結(jié)合。進一步測得能使7或70nm納米金粒徑增大最多、紅移最多的人肝微粒體的臨界濃度。由此推測人肝微粒體的膜結(jié)構(gòu)能在納米金表面形成微囊,從而影響了膜的完整性,導(dǎo)致固定在膜上面的CYPs酶活性的改變。
[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.
【學(xué)位授予單位】：湖南師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：TB383.1;R96

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