臺灣博碩士論文加值系統

過去研究指出，暴露微粒污染物會引起肺部細胞氧化壓力增加，進一步引發發炎反應，然而大氣微粒組成複雜，究竟是何種成分引起細胞毒性有待釐清。過去研究發現細胞暴露在超細粒徑微粒或含有多種組成的汽機車燃燒微粒都能引起細胞產生氧化性物種（Reactive Oxygen Species, ROS），造成氧化壓力的上升。然而人類肺部表面具有多種抗氧化成分的上皮內襯液體（epithelial lining fluid; ELF），能與進入呼吸道的微粒污染物反應減少細胞氧化傷害，但其影響機制並不清楚。本研究目的為探討肺部上皮內襯液體對於各種微粒污染物成分引起細胞氧化傷害的影響。
我們以非細胞系統及細胞系統進行暴露實驗並參考Guobin等人的研究，配製人類肺部上皮內襯液體進行實驗。在非細胞系統中進行各種微粒污染物暴露後的ROS量測，而在細胞系統中利用轉移盤（transwell）進行細胞實驗，在transwell上添加人類肺部上皮內襯液體與肺泡上皮細胞（A-549），測量各種微粒成分與細胞反應後產生之ROS、評估細胞DNA單股斷裂情形及發炎前趨物IL-8量測。微粒濃度方面，選用能產生ROS的最小濃度進行實驗，在超細粒徑碳黑微粒（ufCB）0, 50, 150�慊/ml、過渡金屬（FeSO4）0, 100, 500�嵱及柴油引擎有機萃取物（Diesel Exhaust Particles extract, DEP extract）0, 25, 50�慊/ml下進行4小時的暴露。使用DCFH assay量測ROS、彗星分析法評估細胞DNA單股斷裂及酵素免疫法量測發炎前趨物IL-8。
研究結果顯示，在非細胞系統中，暴露ufCB及FeSO4各種濃度所產生的ROS有隨暴露濃度及暴露時間增加呈現上升的趨勢。此外，在添加ELF的環境下暴露ufCB、FeSO4及 DEP萃取物後，ROS顯著降低（p<0.05），扣除背景值的ROS後，相較於未添加ELF系統的暴露下平均分別減少100％、56.5％及1％。在細胞實驗部分，暴露ufCB、FeSO4及DEP萃取物後，添加ELF於細胞表面能顯著減少氧化性物種產生，相較於未添加ELF系統而言，分別減少91％、83％及1％。在DNA單股斷裂方面，發現FeSO4及 DEP萃取物暴露皆能較控制組引起顯著的DNA單股斷裂，在高濃度暴露下DNA單股斷裂分別較控制組增加67％及23％，然而暴露ufCB的效應並不明顯。此外，添加ELF的ufCB、FeSO4及 DEP萃取物的暴露並不會造成DNA單股斷裂。細胞激素IL-8的分泌在各微粒中並無顯著差異。
研究結果指出，在非細胞或細胞暴露系統中，ufCB、FeSO4及DEP萃取物皆能造成ROS的產生，增加的比率以ufCB最大。此外，添加ELF能有效減少暴露後產生的ROS，減少的比率也以ufCB最大。此外ELF還能進一步保護肺泡上皮細胞免於氧化壓力的傷害。

Previous studies have reported that exposure to particulate matter（PM）induces oxidative stress and respiratory tract inflammation. The component of PM responsible for this health effect remained unclear. Pulmonary epithelial lining fluid（ELF）consists of various antioxidants, which can protect lung cells against the oxidative damage of PM. However the exact mechanism remains unclear. The aim of this study was to determine the effect of ELF on PM-induced oxidative damage.
This study was conducted with and without epithelial cells. We also use synthetic human epithelial lining fluids (ELF, Guobin et al., 1999.) in both cell and cell free system. In cell free system, total ROS was measured with DCFH assay after exposure to ultrafine carbon black (ufCB; at 0, 50, 150ug/ml), transitional metal (FeSO4; at 0,100, 500uM) and organic component（DEP extract; at 0, 25, 50ug/ml）. Subsequently, we established an in vitro transwells exposure system consisting with or without of ELF. A549 cells were exposed to particles for four hours. ROS was then measured using DCFH assay and DNA breakage was determined by single-cell gel electrophoreses (Comet assay).
The results indicated that in cell free system, the amounts of ROS increased with exposure concentration and exposure time. ELF significantly decreased ROS by 100% 、56.5% and 1% as compared to culture medium after ufCB、FeSO4 or DEP extract exposure（p<0.05）. Furthermore, in cell system, ROS decreased 91%、 83%and 1% with the treatment of ELF compared to culture medium after cells exposed to ufCB 、FeSO4 and DEP extract. ELF also can decrease DNA single-strand breakage. Furthermore, there was no difference on IL-8 secretion after exposure to different componemts.
Our results suggest that ELF can decrease total ROS induced by ultrafine carbon black、FeSO4 and DEP extract. Therefore ELF can protect A549 cells from oxidative damage.

摘 要 i
Abstract iii
第一章 前言 8
第二章 文獻回顧 10
2.1微粒的健康效應 10
2.2懸浮微粒的毒性來源 11
2.3微粒毒性與氧化壓力 13
2.3.1 反應性含氧物種與氧化壓力 13
2.3.2微粒毒性與氧化壓力：超細粒徑微粒、過渡金屬及有機物 14
2.3.3其他與ROS的相關研究 16
2.4 肺部抗氧化防禦機制 17
2.4.1肺部上皮內襯液體（ELF） 17
2.4.2微粒污染物與ELF的相關研究 19
2.5氧化傷害效應指標 20
2.5.1發炎反應 20
2.5.2 DNA斷裂與慧星分析 21
第三章 材料與方法 24
3.1 實驗流程 24
3.2 配製ELF.（epithelial lining fluid） 25
3.3微粒、金屬溶液及DEP萃取物配製 25
3.4細胞培養 26
3.5細胞計數及存活率分析 27
3.6微粒、過渡金屬及DEP萃取物暴露 27
3.7 氧化自由基分析 28
3.8 DNA單股斷裂分析 30
3.9 Interleukin-8 分析 31
3.10統計分析 31
第四章 實驗結果 32
4.1氣懸微粒成分分析 32
4.2 暴露時間、濃度與ROS之關係 32
4.3 細胞存活率 32
4.4 ufCB、FeSO4及DEP萃取物暴露對ROS影響 32
4-5 添加ELF對於ufCB、FeSO4及DEP萃取物暴露產生之ROS影響 33
4.6氣懸微粒或ufCB 、FeSO4共同暴露對於細胞ROS的影響 33
4.7不同暴露系統對於ufCB、FeSO4及DEPextract暴露產生DNA單股斷裂影響 34
4.8細胞激素的測量結果 34
第五章 討論與結論 35
5.1 微粒成分與細胞存活率 35
5.2 ufCB、FeSO4及DEP萃取物的ROS 36
5.3 暴露ufCB、FeSO4及DEP萃取物後對於 DNA單股斷裂的影響 38
5.4 ELF對於暴露ufCB、FeSO4及DEP萃取物後產生ROS之影響 38
5.5 ELF對暴露ufCB、FeSO4及DEP萃取物後引起DNA單股斷裂之影響 39
5.6 ROS與細胞激素IL-8之影響 39
5.7 細胞外ROS與DNA單股斷裂之相關 41
5.8 影響ROS量測的因素 41
第六章 參考文獻 44

圖表目錄

表1 ELF的組要成分及濃度 56
表2 暴露大氣微粒所含元素成分及濃度 57
表3 各種暴露狀況下的細胞存活率 58
表4 不同暴露系統中有無細胞對於螢光強度值的影響 59
表5 DEP 有機萃取物對於細胞及非細胞系統中螢光強度值的影響 60
表6 添加ELF能減少ROS的百分比 61
表7 不同暴露狀況下彗星分析結果 62
表8 不同暴露狀況下對於細胞激素IL-8的影響 63
圖1 氧化物新陳代謝 19
圖2 實驗流程圖 24
圖3散射螢光值與H2O2濃度之檢量線 64
圖4-1 FeSO4配置時間與螢光值之關係 65
圖4-2 超細粒徑碳黑微粒（ufCB）配置時間與螢光值之關係 65
圖5 暴露四小時後A-549細胞存活率 66
圖6 有無添加ELF對於暴露過渡金屬FeSO4 後ROS之影響 67
(a) 非細胞系統
(b) 細胞系統
圖7 有無添加ELF對於暴露超細粒徑碳黑微粒ROS之影響 68
(a) 非細胞系統
(b) 細胞系統
圖8有無添加ELF對於DEP萃取物暴露與螢光值之影響 69
(a) 非細胞系統
(b) 細胞系統
圖9 細胞共同暴露FeSO4及ufCB對於螢光值之影響 70
圖10 細胞暴露大氣微粒4小時後螢光值之比較 71
圖11 慧星分析結果;暴露FeSO4溶液4小時後，DNA單股斷裂情形72
圖12 慧星分析結果;暴露ufCB 4小時後，DNA單股斷裂情形 73
圖13 慧星分析結果，添加ELF對於暴露FeSO4後DNA單股斷裂情形74
圖14 慧星分析結果，添加細胞培養液或ELF對於細胞暴露DEP萃取物4小時後DNA單股斷裂的情形 75
圖15 不同暴露物其非細胞ROS值與DNA單股斷裂之相關 76
圖16非細胞ROS值與DNA單股斷裂之相關 77
附錄一 ELF中各種抗氧化酵素之代謝方程式 78
附錄二 細胞內抗氧化防禦能力示意圖 79
附錄三 細胞實驗使用的暴露系統示意圖 80
附錄四 彗星分析影像 81

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