發(fā)布時間：2018-05-05 05:19

  本文選題：芳香烴受體 + 納米顆粒��； 參考：《山東大學》2017年碩士論文

【摘要】：納米材料由于其特異的理化性質已經(jīng)被廣泛應用于諸如航空航天、汽車工業(yè)、環(huán)保建材、體育用品、電子器件以及日常消費品等領域,而且在生物醫(yī)藥領域如診斷、生物成像、生物傳感器和藥物運輸?shù)纫簿哂袕V泛的應用前景。因此人們暴露在納米環(huán)境中的機會也越來越頻繁。相對于從日常產(chǎn)品的接觸中暴露納米顆粒,應用于醫(yī)藥領域的納米顆粒由于可以通過攝入和注射等方式直接進入人體,因此人們更加關注納米顆粒在這一領域額應用的安全性。芳香烴受體(Aryl hydrocarbon receptor,AhR)作為調控外源毒性化合物代謝基因的關鍵核受體,其下游被研究最多也是最經(jīng)典的靶基因是細胞色素P4501A1(CYP1A1)。近期的研究發(fā)現(xiàn)AhR在正常的生理條件下依然發(fā)揮著重要的作用,比如細胞增殖和分化,免疫平衡的調控等對AhR及其相關信號通路的干擾對細胞和生物體正常的生理功能可能產(chǎn)生不利影響。以往對AhR干擾物的研究往往聚焦于小分子化合物,忽略了一系列新穎的人工合成材料如納米顆粒也可能干擾AhR信號通路。已有的研究表明納米顆�？赡軙�干擾CYP1A1等AhR下游基因的表達活性�？紤]到納米顆粒在環(huán)境中的廣泛分布以及對人類健康的高暴露風險,AhR信號通路是否能被納米顆粒干擾是個急需解決的問題。我們首先選擇以穩(wěn)轉熒光素酶報告基因的小鼠肝癌細胞CBG2.8D為實驗模型,評價了幾種常見納米顆粒包括碳納米管(MWCNTs-COOH)、氧化石墨烯(GO-PEG)、納米二氧化硅顆粒(nSiO_2)、納米二氧化鈦顆粒(nTiO_2)、納米金顆粒(AuNPs)以及納米氧化鋅顆粒(nZnO)對AhR信號通路的干擾能力。實驗結果表明上述納米顆粒除nTiO_2外,都具有不同程度干擾AhR-Luciferase通路的能力。為了進一步闡明了納米顆粒與AhR信號通路之間相互作用的內在分子機制,隨后我們選擇了 MWCNTs-COOH為模型,利用實時熒光定量PCR和蛋白質免疫印跡等技術,在基因和蛋白水平驗證了 MWCNTs-COOH對AhR信號通路的干擾作用機制。結果表明MWCNTs-COOH能夠在基因和蛋白水平抑制CYP1A1的表達,同時能夠改變AhR的基因表達水平和其總蛋白含量。但這種現(xiàn)象不是由于ROS所引起。隨后的激酶抑制劑實驗表明MWCNTs-COOH對AhR-CYP1A1信號通路的抑制至少部分是由于胞外調節(jié)蛋白激酶(Extracellular signal-regulated kinase,ERK)介導。而內吞抑制劑實驗同時也表明,對AhR-CYP1A1信號通路的抑制部分需要細胞對MWCNTs-COOH的攝入。本研究不僅對闡明納米顆粒與AhR之間相互作用及其內在機制具有理論意義,而且能夠完善納米顆粒的風險評估,對納米顆粒的安全性設計提供理論依據(jù)。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R114;TB383.1

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