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研究生：	陳姿羽 Chen, Zi-Yu
論文名稱：	探討奈米銀粒子於果蠅模式中誘發的氧化壓力所造成之細胞毒性機轉 Silver nanoparticles (AgNPs) induce ROS-mediated cellular damages in Drosophila
指導教授：	顏賢章 Yan, Shian-Jang
學位類別：	碩士 Master
系所名稱：	醫學院 - 生理學研究所 Department of Physiology
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	43
中文關鍵詞：	奈米銀、氧化壓力
外文關鍵詞：	Silver nanoparticles, oxidative stress
相關次數：	點閱：131 下載：5
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近幾年奈米銀 ( Silver nanoparticles, AgNPs ) 大量應用於各類不同的產品中，如醫療和工業產業之中，這意味著這將增加人類暴露奈米銀的風險。最近研究已顯示奈米銀會活化許多細胞毒性途徑，但是我們目前仍不清楚奈米銀在生物體內是藉由何種分子以及細胞機轉導致毒性，因此我們使用了果蠅（Drosophila）作為體內模式系統來探討奈米銀導致細胞損壞的機轉。我們證實透果蠅的幼蟲透過飲食暴露奈米銀後，會增加幼蟲與蛹的死亡率，並減少成蟲的羽化率。機轉上，我們發現奈米銀會誘導大量的活性氧化物 ( Reactive oxygen species )的產生及劑量相關地活化Nrf2 抗氧化訊號路徑，除此之外亦發現奈米銀會活化細胞自噬作用 (Autophagy)，DNA 損傷( DNA damage )以及細胞凋亡 (Apoptosis )。此外，幼蟲暴露奈米銀也會劑量濃相關地減少成蟲果蠅的壽命。有趣的事是， Nrf2 抗氧化訊號路徑下游基因 superoxide dismutase (SOD)和catalase　(CAT)表現量在幼蟲期暴露過奈米銀的年輕果蠅成蟲會上升，但年老果蠅卻下降的。綜合以上我們已證明奈米銀暴露後會導致各種ROS 介導的細胞毒性效應，並透過降低抗氧化能力對健康/壽命產生長期不利的影響。我們的研究提供奈米銀導致的毒性機轉，且可對奈米銀使用上的安全管理策略之建立提供學理根基。

The growing applications of silver nanoparticles (AgNPs) in various aspects of daily life, such as healthcare and industrial sectors, have raised potential safety and health concerns. Recent evidence has shown that AgNPs induce a variety of cytotoxic pathways. However, the molecular and cellular mechanisms by which AgNPs exert toxicity, particularly in vivo, remain elusive. Here, we used Drosophila as a model system to study mechanisms of AgNPs-induced cellular damages in vivo. We demonstrated that dietary exposure at the larval stage increased larval/pupal lethality, and decreased successful eclosion rates. Mechanically, AgNPs strongly induced reactive oxygen species (ROS) and dose-dependently activated the Nrf2 antioxidant signaling pathway. Moreover, AgNPs dramatically led to activation of autophagy, DNA damage and apoptosis at the larval stage. Furthermore, larval exposure to AgNPs dose-dependently decreased lifespan of adult flies. Interestingly, expression of superoxide dismutase and catalase, downstream target genes of Nrf2 signaling, was decreased in aged but not in young flies. Taken together, AgNPs exert a variety of ROS-mediated cytotoxic effects, and induce long-term adverse effects on health/longevity by decreasing antioxidant capability. Our findings provide insight into mechanisms of AgNPs-induced toxicity, and may provide the scientific foundation for establishing safety management strategies of AgNPs usage.

中文摘要...............................................................................................................................I
Abstract ...............................................................................................................................II
Acknowledgments..............................................................................................................III
Content............................................................................................................................... IV
Table index.......................................................................................................................VII
Figure index ....................................................................................................................VIII
Abbreviations.....................................................................................................................IX
Introduction......................................................................................................................1
1 Applications of AgNPs and their potential health risks....................................................1
2 Toxicity of AgNPs……………………............................................................................2
2.1 Exposure routes……………….....................................................................................2
3 Molecular mechanisms of AgNPs………………………...…………………………..3
3.1 Cellular uptake dictates AgNPs toxicity.......................................................................3
3.2 Oxidative stress as an importance cause of AgNPs toxicity………………...………..3
3.2.1 Imbalance of ROS results in significant cellular damage…………………..………3
3.2.2 AgNPs exposure induces ROS………………………..……………………...……..4
3.3 AgNPs exposure leads to defective autophagy……………………………………. 4
4. Conclusion:......................................................................................................................5
Materials and Methods....................................................................................................7
1 Drosophila strains………….…………………………………………………………7
2 Dietary exposure of Drosophila larvae to AgNPs as a model for investigating their toxicity…………………………............……………………………………....……………7
3 Dihydroethidium (DHE) staining………….....................................................................8
4 Whole-mount immunofluorescence staining……….…………………………….….…8
5 Western blotting……………………………….………………………………..………9
6 Quantitative real-time PCR analysis................................................................................9
7 Longevity assay……......................................................................................................10
8 Paraquat challenge assay................................................................................................10
9 Cncc/Nrf2 reporter gene induction assay ......................................................................10
Results.............................................................................................................................12
1 Dietary administration of AgNPs increased larval/pupal lethality, and decreased eclosion rate.……………………………………………………………………...………..12
2 AgNPs increase ROS level…………..…………….…………………………………..12
3 AgNPs activate Nrf2-dependent antioxidant responses.................................................13
4 AgNPs lead to DNA damage.........................................................................................14
5 AgNPs cause activation of autophagy............................................................................14
6 AgNPs cause apoptosis……………………………...………………………………15
7 Catalase rescues flies from AgNPs-induced toxicity.....................................................15
8 ROS aggravate AgNPs-induced toxicity……………………........................................16
9 AgNPs decrease lifespan................................................................................................16
10 Low concentrations of AgNPs decrease expression levels of antioxidant genes in aged animals. ...............................................................................................................................17
11 AgNPs decrease oxidative stress tolerance in aged flies .............................................18
Discussion........................................................................................................................19
1 Summary........................................................................................................................19
2 Nano-sized Ag particles demonstrate stronger toxic effects due to higher level of bioaccumulation……...………….…………………………………………………........19
3 Autophagy potentially accounts for AgNPs-induced cytotoxicity…………………...20
4 Catalase plays an important role in counteracting AgNPs toxicity................................21
5 Epigenetic regulation and protein corona formation account for reduced ROS resistance capability of AgNPs-exposed flies ……………….………...………..…...........22
References......................................................................................................................23
Table …………………………………………………………………………………27
Figures………………………………………………………………………………….28

                                    

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