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研究生:高瑄伻
論文名稱:臭氧和氣懸微粒對人類上皮細胞DNA傷害之研究
論文名稱(外文):DNA damage in human epithelial cell induced by ozone and particulate matters
指導教授:鄭尊仁鄭尊仁引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:職業醫學與工業衛生研究所
學門:醫藥衛生學門
學類:公共衛生學類
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:62
中文關鍵詞:A5498-oxoguanine彗星試驗Fpg微粒臭氧
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臭氧及氣懸微粒是台灣地區主要的空氣污染物,而過去流行病學研究指出此兩種污染物的作用會導致肺癌,但此兩種污染物所導致的DNA傷害卻不是很清楚。因此本研究探討人類肺部上皮細胞單獨及共同暴露臭氧及氣懸微粒造成DNA傷害情形。利用A549細胞株進行臭氧0、60、80、120 ppb各1小時及氣懸微粒 (PM2.5) 0、50、100 µg/ml各4小時分別及共同暴露,以8-oxoguanine及DNA斷裂作為評估DNA傷害的指標。利用流氏細胞儀偵測其暴露後產生之8-oxoguanine;彗星試驗評估DNA斷裂情形,並且進一步加入Formamidipyrimidine glycosylase (Fpg)氧化壓力修補酵素,以提高偵測氧化壓力的敏感度。結果顯示臭氧暴露之DNA斷裂結果,尾端DNA佔全部DNA亮度的百分比隨暴露濃度上升沒有顯著增加;但加入Fpg修補酵素後,尾端DNA佔全部DNA亮度百分比,較高暴露組 (80、120 ppb) 與控制組相比,達統計上顯著差異 (p=0.03;0.03)。另外偵測8-oxoguanine結果顯示,暴露80、120 ppb與控制組比較亦有顯著差異 (p<0.05)。同樣的,微粒暴露下造成DNA斷裂沒有顯著差異,但在Fpg加入實驗中,則呈顯著差別;而8-oxoguanine則在100 µg/ml下與控制組有顯著差異 (p<0.05)。至於共同暴露120 ppb臭氧及100 µg/ml PM2.5微粒下所造成之DNA傷害則沒有看出加成效應。若利用抗氧化劑:維他命C、E可在50 µM下顯著抑制臭氧120 ppb所產生之8-oxoguanine。
本研究顯示Fpg酵素可增加彗星試驗對氧化壓力偵測敏感度,而臭氧80 ppb一小時的暴露即會造成顯著氧化壓力造成之DNA傷害,本研究結果可作為法規制訂的參考
Particulate matter (PM) and Ozone (O3) are major air pollutants in Taiwan. Previous epidemiological studies have reported that these pollutants are associated with respiratory cancer, but their carcinogenic mechanism is not clear. This study investigated DNA damage induced by ozone and particulate matters alone and in combination on human lung epithelial cell. A549 cell line was exposed to ozone (0、60、80、120 ppb) for 1 hour and PM2.5 (0、50、100 µg/ml) for 4 hours alone and in combination. We used 8-oxoguanine and DNA breakage to evaluate the effects of ozone and particles. 8-oxoguanine was measured by flow cytometry and comet assay was used to assess DNA breakage. Moreover, formamidipyrimidine glycosylase (Fpg) repair enzyme was added to increase the power of detecting oxidative damage. Our study revealed that after ozone exposure, percentage of tail intensity was not significantly associated with exposure concentration. After-adding Fpg repair enzyme, percentage of tail intensity in higher exposure group (80、120 ppb) was significantly higher than control group (0 ppb) (p=0.03 and 0.03, respectively). Furthermore, 8-oxoguanine levels in exposure group (80、120 ppb) were also significantly higher, as compared control group. After particulate matter exposure, percentage of tail intensity was also not significantly associated with exposure concentration. 8-oxoguanine levels in 100 µg/ml were significantly higher than that in control group (p<0.05). As to co-exposure, the combination effects in DNA damage did not show an additive effect. Futher, the antioxidants, vitamin C and vitamin E could significantly inhibit 8-oxoguanine induced by ozone exposure of 120ppb.
This study reveals adding Fpg enzyme could increase the sensitivity of comet assay for detecting oxidative stress. Importantly, ozone 80 ppb exposure for 1 hour could induce significant DNA damage. Thus, our finding may be useful in standard setti
摘要 i
Abstract ii
目錄 iii
圖表目錄 v
第一章 前言 6
1.1台灣空氣污染現況 6
1.2臭氧與氣懸微粒流行病學研究 6
1.3臭氧與氣懸微粒與氧化壓力相關研究 7
1.4氧化壓力與DNA傷害指標 8
第二章 文獻回顧 10
2.1潛在致癌性相關研究 10
2.2臭氧與氧化壓力 11
2.3氣懸微粒與氧化壓力 12
2.4反應性氧化產物與DNA傷害 13
2.5 DNA傷害指標-氧化鹼基 14
2.6 DNA傷害指標-DNA斷裂 16
第三章 材料與方法 19
3.1微粒採樣收集 19
3.2微粒元素分析及微粒懸浮液處理分析 19
3.3細胞培養 20
3.4細胞存活率分析 20
3.5微粒暴露實驗 21
3.6臭氧暴露實驗 21
3.7臭氧及微粒共同暴露 22
3.8抗氧化劑添加實驗 23
3.9 8-oxoguanine分析 24
3.10 DNA斷裂分析 25
3.11統計分析 26
第四章 研究結果 27
4.1氣懸微粒成分分析 27
4.2細胞存活率 27
4.3臭氧和微粒分別暴露對細胞產生DNA斷裂影響 27
4.4臭氧和微粒分別暴露對細胞產生8-oxoguanine影響 28
4.5臭氧及微粒共同暴露對細胞產生DNA傷害影響 28
4.6抗氧化劑對臭氧抑制8-oxoguanine之效應 28
第五章 討論 30
第六章 參考文獻 36
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