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研究生:薛翔予
研究生(外文):Hsiang-Yu Hsueh
論文名稱:奈米銀粒子誘導CL5肺腺癌細胞株細胞激素表現及環境毒物之生物效應
論文名稱(外文):Induction of cytokines by silver nanoparticles in CL5 human lung adenocarcinoma cells and biological effects of environmental toxicants
指導教授:翁祖輝翁祖輝引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:毒理學研究所
學門:醫藥衛生學門
學類:其他醫藥衛生學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:106
中文關鍵詞:奈米銀粒子愛殺松機車廢氣纖維母細胞生長因子 9白細胞介素 1β細胞色素 P450
外文關鍵詞:silver nanoparticlesethionmotorcycle exhaustFGF-9IL-1βCYP
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本篇論文主要目的為探討:一、奈米銀粒子對肺腺癌細胞株 CL5 細胞激素表現之影響;二、農藥愛殺松對雄性大鼠肝臟細胞色素 P450 表現之影響;三、機車廢氣對雄性大鼠及其子代肝臟細胞色素 P450 表現之影響。近年來,奈米銀粒子已經被應用於生物科技以及生命科學領域,奈米物質的粒徑為1到100 nm,這種粒徑的銀粒子可能具有特殊的物理、化學以及毒物動力學性質。本篇主要探討不同粒徑大小的奈米級的銀粒子Ag (7 nm)、Ag (50 nm) 對肺腺癌細胞 CL5 mRNA表現的影響,從 reverse transcriptase-polymerase chain reaction (RT-PCR) 分析結果顯示,Ag (7 nm) 會增加 fibroblast growth factor (FGF)-9 以及 interleukin (IL)-1β 的 mRNA 表現; Ag (50 nm) 增加 IL-1β 的 mRNA 表現,而 FGF-9 則沒有影響;AgNO3 會增加 FGF-9 mRNA 的表現,而 IL-1β 則沒有影響。在酵素部份 Ag (7 nm)、AgNO3 會使 ethoxyrcoumarin O-deethylase (ECOD) 活性、peroxide production 上升,glutathione S-transferase (GST) 活性與 total glutathione content 下降,lipid peroxidation (LPO) 沒有影響;Ag (50 nm) 使 ECOD 活性上升,而 GST、total glutathione content、LPO 則沒有影響;AgNO3 會使 ECOD 活性、LPO 上升,GST、total glutathione (GSH) content 下降。低粒徑的奈米粒子會對 CL5 細胞造成較強的氧化性傷害,高粒徑則較弱,對 FGF-9 與 IL-1β 的 mRNA 誘導表現也較強,氧化性傷害與 peroxide 的生成有關。
愛殺松是屬於有機磷類神經毒性殺蟲,用於防治蚜蟲、蝨、介殼蟲、葉蟬、蛆等病蟲害,它可以使用於許多種類的食物、纖維、裝飾類的作物,以及溫室作物。本研究的主要目的試探討愛殺松在雄性大鼠肝臟細胞色素 P450 的表現,分為低劑量與高劑量兩個部份,雄性大鼠以腹腔注射愛殺松,低劑量處理為 1、10 mg/kg;高劑量處理為 20 mg/kg,1 天 1 次進行 4 天,於肝臟微粒體,低劑量組 10 mg/kg 愛殺松使 pentoxyresorufin O-dealkylase (PROD) 活性增加,cytochrome P450、NADPH -cytochrome P450 reductase、methoxyresorufin O-demethylase 與 aniline hydroxylation 則是下降;高劑量組 20 mg/kg 愛殺松使 PROD 活性上升。在西方墨點法和 RT-PCR 分析中 CYP2B 的蛋白量和 mRNA 都有增加,且具有劑量效應關係。研究結果發現愛殺松是雄性大鼠肝臟內 CYP2B 的誘導物。
機車在台灣是最主要的交通工具之一,其排放的廢氣造成嚴重的空氣汙染,因此廢氣的為害對於人體的影響是我們所必須重視的。本實驗中藉由觀察大鼠肝臟內 P450 mRNA 的改變來了解機車廢氣對生物體及其後代的影響。實驗結果顯示雄性大鼠肝臟內的 CYP1A1、CYP2B、CYP2E1 mRNA 表現有上升,CYP2C12 則是下降,這顯示機車廢氣會對肝臟內的 P450 mRNA 產生影響。暴露機車廢氣的雄性大鼠與未暴露的雌性大鼠交配之後產下了第二代,第二代雄性大鼠肝臟內的 CYP1A2、CYP3A mRNA 表現於出生後 56、98 天時有下降,140 天則沒有影響;第二代雌性大鼠肝臟內的 CYP1A2 mRNA 於出生後 56 天時有下降,98、140 天時則沒有影響,CYP3A1 mRNA 於56、98 天時有下降,140 天沒有影響;CYP2B、CYP2C11 mRNA 只有雄性子代於 98 天時有下降。研究結果發現機車廢氣對雄性大鼠及其子代肝臟 P450 mRNA 表現有影響,因此應進一步針對其機制做探討。
The major purposes of these studies were to determine the effects of silver nanoparticles on cytokine expression in humane lung adenocarcinoma cells CL5. the ability of ethion to induce cytochrome P450 expression in male rat liver, and the effect of paternal exposure to motorcycle exhaust on cytochrome P450 expression in rat liver.
Nanoparticles are applied to the domains of life science and biotechnology. The diameter of nanoparticle is 1 to 100 nm, so it may have special physics and toxicokinetic properties. The major objective of this study was to investigate the effects of different scale diameter nanoparticles on mRNA expression in human lung adenocarcinoma cells CL5. The result of reverse transcriptase-polymerase chain reaction (RT-PCR) analysis showed that Ag (7 nm) increased fibroblast growth factor (FGF)-9 and interleukin (IL)-1β mRNA expression. Ag (50 nm) increased IL-1β mRNA expression but no effect on FGF-9. AgNO3 increased FGF-9 mRNA expression but had no effects on IL-1β. Ag (7 nm) and AgNO3 increased ethoxyrcoumarin O-deethylase (ECOD) activity and peroxide production, decreased glutathione S-transferase (GST) activity and total glutathione content, had no effects on lipid peroxidation. Ag (50 nm) increased ECOD activity but had no effects on GST activity, total glutathione content, and lipidperoxidation. The present finding shows that the smaller diameter nanoparticles cause more oxidative damage and FGF-9 and IL-1β mRNA expression than the larger diameter one did. Peroxide production may cause oxidative damage.
Ethion is an organophosphate pesticide used to kill aphids, mites, leafhoppers, and maggots . It is used on a wide variety of food, fiber, ornamental crops, and greenhouse crops. The major objective of the present study was to investigate the ability of ethion to modulate CYP-dependent monooxygenase in rat liver. Male rats were treated with 1, 10, and 20 mg/kg ethion intraperitoneally once daily for 4 days. In liver microsome, 10 mg/kg ethion treatment increased pentoxyresorufin O-dealkylase (PROD) activity but decreased cytochrome P450 content, NADPH -cytochrome P450 reductase, methoxyresorufin O-demethylase activity and aniline hydroxylase activity. Treatment with 20 mg/kg ethion increased PROD activity. The result of immunoblot and RT-PCR analyses showed that ethion dose dependently induced CYP2B protein and mRNA in the liver. The present finding shows that ethion is an inducer of CYP2B in male rat liver .
Motorcycle is a popular means of transportation in Taiwan. The exhaust causes air pollution seriously and human expose to the exhaust is an important health issue. Thus, we have studied the effect of motorcycle exhaust on cytochrome P450 mRNA in male rats and its offspring. Motorcycle exhaust increased CYP1A1, CYP2B and CYP2E1 mRNA expression but decreased CYP2C11 mRNA expression in the liver. Motorcycle exhaust-exposed male rats were mated with untreated female rats and pregnant female rats were allowed to deliver. In the F1 generation, CYP1A2 and CYP3A mRNA expression was decreased in male rats liver on postnatal day (PND) 56 and 98 but no effect was observed on 140. CYP1A2 and mRNA expression was decreased in female offspring liver on PND 56 but no effects were observed on PND 98 and 140. CYP3A1 mRNA expression was decreased in female offspring liver on PND 56 and 98 but not on PND 140. CYP2B and CYP2C11 mRNA was only decreased in male rat liver on PND 98. The present finding shows that motorcycle exhaust changes P450 mRNA expression in male rats and offspring liver, so we should find out the mechanism in the future.
中文摘要 1
Abstract 3

第一部分 奈米銀粒子對肺腺癌纖維母細胞株細胞激素之影響
前言 5
第一節、奈米粒子的使用概況 5
第二節、FGF-9 (fibroblast growth factors -9) 介紹 6
第三節、細胞激素的介紹 6
第四節、生物體內的細胞色素 P450 及其改變的毒理意義 7
材料與方法 9
第一節、實驗材料 9
第二節、實驗方法 9
結果 15
第一節、奈米銀粒子對婦女肺腺癌 CL5 細胞株細胞毒性之影響 15
第二節、奈米銀粒子對 CL5 之生長因子 FGF-9 mRNA 誘導濃度效應關係 15
第三節、奈米銀粒子對 CL5 之生長因子 FGF-9 mRNA 誘導時間效應關係 15
第四節、奈米銀粒子對 CL5 之 IL-1β mRNA 誘導濃度效應關係 16
第五節、奈米銀粒子對 IL-1β 誘導時間效應關係 16
第六節、AgNO3 對 CL5 之生長因子 FGF-9 與 IL-1β mRNA 誘導濃度效應關係 16
第七節、AgNO3 對 CL5 之生長因子 FGF-9 與 IL-1β mRNA 誘導時間效應關係 16
第八節、以real-time RT-PCR 確認 standard RT-PCR 分析結果 17
第九節、奈米銀粒子對 CYP1A1、CYP1B1、FGF-2、VEGF-C mRNA 誘導濃度效應關係 17
第十節、奈米銀粒子對 IL-1α、IL-6、cyclooxygenase (COX)-2、tumor necrosis factor (TNF)-α mRNA 誘導濃度效應關係 17
第十一節、N-acetylcysteine (NAC) 對Ag (7 nm) 誘導CL5 生長因子 FGF-9 與 IL-1β mRNA 表現之影響 17
第十二節、奈米銀粒子對 CL5 細胞過氧化物生成時間效應關係 18
第十三節、奈米銀粒子對 CL5 細胞 GSH content 與 lipid peroxidation 的時間效應關係 18
第十四節、奈米銀粒子對 CL5 細胞 ECOD 與 GST 酵素活性的時間效應關係 19
討論 20
第一節、CL5 肺腺癌纖維母細胞株 FGF-9 mRNA 的表現 20
第二節、CL5 肺腺癌纖維母細胞株 IL-1β mRNA 的表現 22
第三節、氧化壓力 22
第四節、結論 24

第二部分 農藥愛殺松對雄性大鼠肝臟 P450 表現之影響
前言 25
第一節、農藥的使用概況 25
第二節、有機磷農藥 25
第三節、愛殺松的物理化學特性及作用 26
第四節、愛殺松的急性毒性 26
第五節、愛殺松的慢性毒性 27
第六節、愛殺松的其他毒性 27
第七節、農藥對 P450 的影響 27
材料與方法 29
第一節、實驗材料 29
第二節、實驗方法 29
結果 32
第一節、農藥愛殺松對雄性大鼠肝臟 P450、細胞色素 b5、含量與 NADPH -cytochrome P450 reductase 活性的影響 32
第二節、農藥愛殺松對雄性大鼠肝臟 EROD、MROD、PROD、aniline hydroxylase 活性之影響 32
第三節、高劑量農藥愛殺松對雄性大鼠肝臟 P450、細胞色素 b5、含量與 NADPH -cytochrome P450 reductase 活性的影響 32
第四節、高劑量農藥愛殺松對雄性大鼠肝臟 EROD、MROD、PROD、aniline hydroxylase 活性之影響 33
第五節、愛殺松對肝臟內的細胞色素 P450 2B 蛋白量的影響 33
第六節、農藥愛殺松對雄性大鼠肝臟細胞色素 P450 2B mRNA 表現之影響 33
討論 34
第一節、愛殺松對肝臟 P450 表現之影響 34
第二節、結論 36

第三部份 機車廢氣對雄性大鼠肝臟及其子代 P450 表現之影響
前言 37
第一節、汽機車造成的空氣汙染 37
第二節、機車廢氣的毒性及所帶來的影響 37
第三節、內分泌干擾物質對 P450 的影響 39
材料與方法 40
第一節、實驗材料與方法 40
結果 41
第一節、機車廢氣對雄性大鼠肝臟細胞色素 P450 1A1、2B、2E1 mRNA 表現之影響 41
第二節、機車廢氣對雄性大鼠肝臟細胞色素 P450 1A2、3A1、2C11、2C12 mRNA 表現之影響 41
第三節、機車廢氣對雄性子代大鼠肝臟細胞色素 P450 1A2 mRNA 表現之影響 41
第四節、機車廢氣對雌性子代大鼠肝臟細胞色素 P450 1A2 mRNA 表現之影響 42
第五節、機車廢氣對雄性子代大鼠肝臟細胞色素 P450 3A1 mRNA 表現之影響 42
第六節、機車廢氣對雌性子代大鼠肝臟細胞色素 P450 3A1 mRNA 表現之影響 42
第七節、機車廢氣對子代大鼠於 PND 56 肝臟細胞色素 P450 2B、2E1、2C11、2C12 mRNA 表現於之影響 42
第八節、機車廢氣對子代大鼠於 PND 98 肝臟細胞色素 P450 2B、2E1、2C11、2C12 mRNA 表現於之影響 42
第九節、機車廢氣對子代大鼠於 PND 140 肝臟細胞色素 P450 2B、2E1、2C11、2C12 mRNA 表現於之影響 43
討論 44
第一節、機車廢氣對親代大鼠 P450 mRNA 表現之影響 44
第二節、機車廢氣對子代大鼠 P450 mRNA 表現之影響 45
第三節、結論 46

總結 47
參考文獻 48
圖表 60



表目錄
Table 1. Concentration-response relationship and time course of effects of Ag nanoparticle on viability of human lung adenocarcinoma CL5 cells 60
Table 2. Effects of Ag nanoparticle on FGF-9 and IL-1 β mRNA in human lung adenocarcinoma CL5 cells analyzed by real-time PT-PCR 61
Table 3. Time courses of effects of Ag nanoparticle on peroxide production in human lung adenocarcinoma CL5 cells 62
Table 4. Concentration-response relationships of effects of Ag (7 nm) on cytokines mRNA in human lung adenocarcinoma CL5 cells 63
Table 5. Concentration-response relationships of effects of Ag (50 nm) on cytokines mRNA in human lung adenocarcinoma CL5 cells 64
Table 6. Time courses of effects of Ag (7 nm), Ag (50 nm), and AgNO3 on cytokines mRNA in human lung adenocarcinoma CL5 Cells 65
Table 7. Time courses of effects of Ag nanoparticle on GSH content and lipid peroxidation in human lung adenocarcinoma CL5 cells 66
Table 8. Time courses of effects of Ag nanoparticle on ECOD and GST activities in human lung adenocarcinoma CL5 cells 67
Table 9. Effects of treatment with ethion on organ weights in rats 68
Table 10. Effects of treatment with ethion on organ/body weight ratios in rats 69
Table 11. Effects of treatment with ethion on monooxygenase acitivities in rat liver 70
Table 12. Effects of treatment with ethion on cytochrome P450, cytochrome b5 contents, and NADPH -cytochrome P450 reductase acitivity in rat liver 71
Table 13. Effects of treatment with ethion on organ weights in rats 72
Table 14. Effects of treatment with ethion on organ/body weight ratios in rats 73
Table 15. Effects of treatment with ethion on monooxygenase acitivities in rat liver 74
Table 16. Effects of treatment with ethion on cytochrome P450, cytochrome b5 contents, and NADPH -cytochrome P450 reductase acitivity in rat liver 75
Table 17. Immunoblot and RT-PCR analyses of cytochrome P450 2B protein and mRNA in the livers from controls and rats treated with ethion 76
Table 18. RT-PCR analyses of cytochromes P450 1A1, 1A2, 2B, 2E1, 2C11, 2C12, and 3A in the livers from controls and male rats treated with motorcycle exhaust 77
Table 19. RT-PCR analyses of cytochromes P450 1A2, 2B, 2E1, 2C11, 2C12, and 3A in the livers from controls and male offspring of rats treated with motorcycle exhaust paternally 78
Table 20. RT-PCR analysis of cytochromes P450 1A2, 2B, 2E1, 2C11, 2C12, and 3A in the livers from controls and female offspring of rats treated with motorcycle exhaust paternally 79
Table 21. Oligonucleotide primers for standard RT-PCR analyses of growth factor, cytokine, cytochrome P450, and β-Actin of human lung adenocarcinoma cell line CL5 80
Table 22. Oligonucleotide primers for standard RT-PCR analyses of cytochrome P450 and cyclophilin of rat 81
Table 23. Oligonucleotide primers for real-time RT-PCR analyses of FGF-9, IL-1β, and β-Actin of human lung adenocarcinoma cell line CL5 82



圖目錄
Fig. 1. Concentration-response relationship of effects of Ag nanoparticles on FGF-9 mRNA in human lung adenocarcinoma CL5 cells 83
Fig. 2. Time course of effects of Ag nanoparticle on FGF-9 mRNA in human lung adenocarcinoma CL5 cells 84
Fig. 3. Concentration-response relationship of effects of Ag nanoparticles on IL-1β mRNA in human lung adenocarcinoma CL5 cells 85
Fig. 4. Time course of effect of Ag nanoparticles on IL-1β mRNA in human lung adenocarcinoma CL5 cells 86
Fig. 5. Concentration-response relationship of effect of AgNO3 on FGF-9 and IL-1β mRNA in human lung adenocarcinoma CL5 cells 87
Fig. 6. Time courses of effects of AgNO3 on FGF-9 and IL-1β mRNA in human lung adenocarcinoma CL5 cells 88
Fig. 7. CYP1A1, CYP1B1, FGF-2, and vascular endothelial growth factor (VEGF)-C mRNA expression in human adenocarcinoma CL5 cells treated with Ag nanoparticles89
Fig. 8. IL-1α, IL-6, COX-2, and TNF-α mRNA expression in human adenocarcinoma CL5 cells treated with Ag nanoparticles 90
Fig. 9. Effects of NAC on induction of FGF-9 and IL-1β mRNA by Ag nanoparticles in human lung adenocarcinoma CL5 cells 91
Fig. 10. Effect of treatment with ethion on cytochrome P450 2B protein of rat liver microsomes analyzed by immunoblot 92
Fig. 11. RT-PCR analysis of cytochrome P450 2B mRNA in the livers from controls and rats treated with ethion 93
Fig. 12. Effect of 2-stoke ME exposure on cytochrome P450 1A1 mRNA in rat liver 94
Fig. 13. Effect of 2-stoke ME exposure on cytochrome P450 1A2 mRNA in rat liver 95
Fig. 14. Effect of 2-stoke ME exposure on cytochrome P450 2B mRNA in rat liver 96
Fig. 15. Effect of 2-stoke ME exposure on cytochrome P450 3A mRNA in rat liver 97
Fig. 16. Effect of 2-stoke ME exposure on cytochrome P450 2E1 mRNA in rat liver 98
Fig. 17. Effect of 2-stoke ME exposure on cytochrome P450 2C11 and 2C12 mRNA in rat liver 99
Fig. 18. RT-PCR analysis of cytochrome P450 1A2 mRNA in the livers from controls and male offspring of rats treated with motorcycle exhaust paternally 100
Fig. 19. RT-PCR analysis of cytochrome P450 3A1 mRNA in the livers from controls and male offspring of rats treated with motorcycle exhaust paternally 101
Fig. 20. RT-PCR analysis of cytochrome P450 1A2 mRNA in the livers from controls and female offspring of rats treated with motorcycle exhaust paternally 102
Fig. 21. RT-PCR analysis of cytochrome P450 3A1 mRNA in the livers from controls and female offspring of rats treated with motorcycle exhaust paternally 103
Fig. 22. RT-PCR analysis of cytochrome P450 2B, 2E1, 2C11, and 2C12 mRNA in the livers from controls and male and female offspring on PND 56 of rats treated with motorcycle exhaust paternally 104
Fig. 23. RT-PCR analyses of cytochromes P450 2B, 2E1, 2C11, and 2C12 mRNA in the livers from controls and male and female offspring on PND 98 of rats treated with motorcycle exhaust paternally 105
Fig. 24. RT-PCR analyses of cytochromes P450 2B, 2E1, 2C11, and 2C12 mRNA in the livers from controls and male and female offspring on PND 140 of rats treated with motorcycle exhaust paternally 106
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