常见纳米颗粒干扰芳香烃受体信号通路机制的初步研究

发布时间：2018-05-05 05:19

本文选题：芳香烃受体 + 纳米颗粒　；参考：《山东大学》2017年硕士论文

【摘要】：纳米材料由于其特异的理化性质已经被广泛应用于诸如航空航天、汽车工业、环保建材、体育用品、电子器件以及日常消费品等领域,而且在生物医药领域如诊断、生物成像、生物传感器和药物运输等也具有广泛的应用前景。因此人们暴露在纳米环境中的机会也越来越频繁。相对于从日常产品的接触中暴露纳米颗粒,应用于医药领域的纳米颗粒由于可以通过摄入和注射等方式直接进入人体,因此人们更加关注纳米颗粒在这一领域额应用的安全性。芳香烃受体(Aryl hydrocarbon receptor,AhR)作为调控外源毒性化合物代谢基因的关键核受体,其下游被研究最多也是最经典的靶基因是细胞色素P4501A1(CYP1A1)。近期的研究发现AhR在正常的生理条件下依然发挥着重要的作用,比如细胞增殖和分化,免疫平衡的调控等对AhR及其相关信号通路的干扰对细胞和生物体正常的生理功能可能产生不利影响。以往对AhR干扰物的研究往往聚焦于小分子化合物,忽略了一系列新颖的人工合成材料如纳米颗粒也可能干扰AhR信号通路。已有的研究表明纳米颗粒可能会干扰CYP1A1等AhR下游基因的表达活性。考虑到纳米颗粒在环境中的广泛分布以及对人类健康的高暴露风险,AhR信号通路是否能被纳米颗粒干扰是个急需解决的问题。我们首先选择以稳转荧光素酶报告基因的小鼠肝癌细胞CBG2.8D为实验模型,评价了几种常见纳米颗粒包括碳纳米管(MWCNTs-COOH)、氧化石墨烯(GO-PEG)、纳米二氧化硅颗粒(nSiO_2)、纳米二氧化钛颗粒(nTiO_2)、纳米金颗粒(AuNPs)以及纳米氧化锌颗粒(nZnO)对AhR信号通路的干扰能力。实验结果表明上述纳米颗粒除nTiO_2外,都具有不同程度干扰AhR-Luciferase通路的能力。为了进一步阐明了纳米颗粒与AhR信号通路之间相互作用的内在分子机制,随后我们选择了 MWCNTs-COOH为模型,利用实时荧光定量PCR和蛋白质免疫印迹等技术,在基因和蛋白水平验证了 MWCNTs-COOH对AhR信号通路的干扰作用机制。结果表明MWCNTs-COOH能够在基因和蛋白水平抑制CYP1A1的表达,同时能够改变AhR的基因表达水平和其总蛋白含量。但这种现象不是由于ROS所引起。随后的激酶抑制剂实验表明MWCNTs-COOH对AhR-CYP1A1信号通路的抑制至少部分是由于胞外调节蛋白激酶(Extracellular signal-regulated kinase,ERK)介导。而内吞抑制剂实验同时也表明,对AhR-CYP1A1信号通路的抑制部分需要细胞对MWCNTs-COOH的摄入。本研究不仅对阐明纳米颗粒与AhR之间相互作用及其内在机制具有理论意义,而且能够完善纳米颗粒的风险评估,对纳米颗粒的安全性设计提供理论依据。
[Abstract]:Nanomaterials have been widely used in such fields as aerospace, automobile industry, environmental protection building materials, sporting goods, electronic devices and consumer goods due to their specific physical and chemical properties, as well as in biomedical fields such as diagnostics, biomedical imaging, etc. Biosensors and drug transportation also have a wide range of applications. As a result, opportunities for exposure to nanoscale environments are becoming more and more frequent. Compared with the exposure of nanoparticles from the daily products, the nanoparticles used in the field of medicine can enter the human body directly through ingestion and injection, so people pay more attention to the safety of nanoparticles in this field. Aryl hydrocarbon receptor AhRis is a key nuclear receptor for regulating the metabolic genes of exogenous toxic compounds. The most studied and classic downstream target gene is cytochrome P4501A1CY CYP1A1. Recent studies have found that AhR still plays an important role in normal physiological conditions, such as cell proliferation and differentiation. The regulation of immune balance may have adverse effects on the normal physiological function of cells and organisms due to the interference of AhR and its related signaling pathways. Previous studies on AhR interferences have focused on small molecular compounds, ignoring a series of novel synthetic materials such as nanoparticles that may interfere with the AhR signal pathway. Previous studies have shown that nanoparticles may interfere with the expression activity of AhR downstream genes such as CYP1A1. Considering the widespread distribution of nanoparticles in the environment and the high exposure risk to human health, whether the AhR signaling pathway can be interfered by nanoparticles is an urgent problem to be solved. We first selected mouse hepatoma cell line CBG2.8D with stable luciferase reporter gene as the experimental model. The interference ability of several common nanoparticles, including MWCNTs-COOHU, graphene oxide GO-PEGN, nano-silica particles, nSiO2O _ 2, TIO _ 2 particles, au _ 2O _ 2, au _ (NPs) and ZnO _ 2O _ 2, to the AhR signal pathway was evaluated. The experimental results show that all the nanoparticles except nTiO_2 have the ability to interfere with the AhR-Luciferase pathway to varying degrees. In order to further elucidate the intrinsic molecular mechanism of interaction between nanoparticles and AhR signaling pathway, we then selected MWCNTs-COOH as a model, using real-time fluorescent quantitative PCR and Western blot techniques. The interference mechanism of MWCNTs-COOH on AhR signaling pathway was verified at the gene and protein levels. The results showed that MWCNTs-COOH could inhibit the expression of CYP1A1 at the gene and protein levels, and change the gene expression level and the total protein content of AhR at the same time. But this phenomenon is not caused by ROS. Subsequent kinase inhibitor experiments showed that the inhibition of AhR-CYP1A1 signaling pathway by MWCNTs-COOH was at least partly mediated by extracellular signal-regulated kinase (ERK). The endocytosis inhibitor experiment also showed that the inhibition of AhR-CYP1A1 signaling pathway required cell uptake of MWCNTs-COOH. This study not only has theoretical significance to clarify the interaction between nanoparticles and AhR and its intrinsic mechanism, but also can improve the risk assessment of nanoparticles and provide theoretical basis for the safety design of nanoparticles.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R114;TB383.1

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