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研究生:劉怡汶
研究生(外文):Yi-Wen Liu
論文名稱:以細胞株直接暴露系統應用於評估燃燒產物細胞毒性
論文名稱(外文):Using cell direct exposure system in evaluating toxicity of combustion products
指導教授:吳佩芝吳佩芝引用關係戴聿彤戴聿彤引用關係
指導教授(外文):Pei-Chih WuYu-Tung Dai
口試委員:蕭大智葉靜華吳佩芝
口試委員(外文):Ta-Chih HsiaoChing-Hua YehPei-Chih Wu
口試日期:2013-07-25
學位類別:碩士
校院名稱:長榮大學
系所名稱:職業安全與衛生學系碩士班
學門:醫藥衛生學門
學類:公共衛生學類
論文種類:學術論文
論文出版年:2013
畢業學年度:102
語文別:中文
論文頁數:50
中文關鍵詞:燃燒產物直接暴露系統氣、液介面細胞株存活率
外文關鍵詞:Burning productDirect exposure systemAir Liquid InterfaceCell viability
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近年來生活方式的改變和空氣污染日益嚴重的狀況,讓我們更重視環境中的污染物對人體的健康危害。空氣污染中,無論是室內或室外都有不同的污染源,環境中的氣膠微粒,其形態與特性至進入體內過程中會因為環境中的許多因素而受到改變,對人體的健康影響也有待研究。
傳統上對微粒毒性的研究,大部分是將微粒捕集並萃取下來,細胞毒理的實驗中證實這些微粒會對細胞造成傷害,但這過程中卻已經改變微粒的特性,近幾年陸續開發各種的暴露系統,也搭配著新型態氣、液介面培養法(Air-Liquid interface, ALI)持續的被運用來評估污染物對細胞的毒理反應,但是各類的暴露系統還能有許多改進空間。本研究使用蕭等人改良的ALI暴露系統搭配燃燒系統進行燃燒產物的細胞毒理測試,次評估ALI暴露系統應用在燃燒產物細胞毒理評估之可行性。
本研究將線香放置燃燒系統中(60x60x60 cm3)使用高壓電源供應器對暴露系統施加電壓,讓微粒直接沉積 Transwell insert 的細胞上,使細胞暴露燃燒產物,進一步使用掃瞄式電移動度粒徑分析儀(SMPS)監測微粒的濃度及系統的穩定度。燃燒系統會產生三組濃度,讓細胞同時暴露混合污染物,再加裝濾網過濾微粒,讓細胞單純暴露氣態污染物,進而比較其毒性差異。暴露完之後會以trypan blue染色法及MTT assay來計算細胞的存活率,並量測細胞釋放ROS的產量評估燃燒產物與細胞氧化性壓力的關係。
研究結果顯示燃燒系統可以長時間的提供一個穩定的產生源,產生源的粒徑分布範圍為100~400nm,且以單峰呈現。數目中位數粒徑隨著產生源不同介於163nm~195nm之間。本暴露系統的沉積率在施加電壓後可以使微粒整體的沉積率提升5%~15%。細胞微粒的暴露劑量與Trypan blue 染色法所呈現細胞的存活率的減少率呈現顯著的線性關係(R2=0.9501)與ROS釋放量的增加率亦呈現顯著的線性關係(R2=0.9071)。暴露氣態污染物的細胞存活率皆高於暴露混合污染物細胞,反應出暴露混合物的毒性高過於暴露氣態污染物。
加裝HEPA濾網可以有效的除去微粒,也呈現暴露混合物的毒性高過於暴露氣態污染物,透過濃度、沉積率與暴露時間間接推估細胞微粒的暴露劑量,不同的暴露劑量對於Trypan blue 染色法所呈現細胞的存活率的減少率及ROS釋放量的增加率都有顯著的相關性,顯示本系統可有效使微粒暴露不同劑量燃燒產物,並可利用Trypan blue及ROS產量評估出混合物及氣態污染物對細胞的毒性危害。但本研究在細胞上的整體沉積率後續仍需以電子顯微鏡確認之。
關鍵字:燃燒產物,直接暴露系統,氣、液介面細胞株,存活率

Change of lifestyle and worsen ambient air pollution, high light the importance of the environmental hazards. Particulate matters both from indoor and outdoor sources with different characteristics in composition and size might cause different adverse effects.
Conventional cell toxicity studies for particles were usually on the filter trapped and extracted for cell exposure .However, the characteristics of particles, such as volatile compounds and size have been changed during the process.
Several types of exposure system with integrating cell culture in air-liquid interface, ALI have developed, to assess a variety of air pollutants, especially air mixtures.
One study use a modified ALI exposure system developed by Hsiao et al with combustion products from the combustion systems for cell toxicity testing. We will evaluate the feasibility of the ALI exposure system using for air mixtures from combustion.
The incense was placed in burning system (60x60x60cm3) and high voltage power supply was applied on the exposure system. To increase particles deposition on Transwell insert. Scanning Mobility Particle Sizer (SMPS) was used to measure the particles levels in the exposure system. There levels of combustion products were introduced to cell exposure chamber. Filtration system was then used to remove the particles from the system, to create a gas phase exposure scenario for ALI. Trypan blue staining and MTT assay were used to measure the cell viability, and ROS production was used to evaluate the oxidative stress from combustion products.
One results shown that burning system can provide a stable 6 hrs source for combustion. The range of particle diameter were 100 to 400 nm with unimodal distribution. The number median diameter were between 163nm to 195nm from three levels of generation source. The deposition rate of the exposure system shows the increased 5% to 20% with applying voltage.
Cell viability reduction rate showed a significant decrease with increasing exposing doses in a linear relationship (R2 = 0.9071). The increase of ROS release also showed a significant linear relationship with exposing doses (R2 = 0.9071). Linear dose dependent effects also shown on gas phase exposure. However, the toxicity were lower than mixtures of combustion.
One study use significantly, de linear dose-dependent effects deposition rate and exposure time to estimate the exposure dose for cells both could be observed on reduction rate of cell viability and the increase of ROS for mixture of combustion and only gas phase. One system can effectively use to different combustion products, showing the overall deposition rate on ALI cells should further examine by electron microscopy in the near future.
Keyword: Burning product, Direct exposure system, Air Liquid Interface,
Cell viability

中文摘要 I
Abstract III
致謝 V
表目錄 IX
圖目錄 X
第一章 前言 1
1.1研究背景 1
1.2研究目的 3
第二章 文獻回顧 4
2.1空氣污染物種類 4
2.1.1氣狀污染物 4
2.1.2粒狀污染物 5
2.1.3 燃燒產物特性 6
2.1.3.1燃燒產物對人體的健康影響 6
2.1.3.2 動物研究 7
2.1.3.3 細胞毒理研究 7
2.2 細胞株直接暴露系統開發 8
2.2.1 傳統細胞毒理評估方法 8
2.2.2細胞株直接暴露系統 8
2.2.3不同暴露系統的設計差別 9
2.2.3.1重力沉降 9
2.2.3.2慣性衝擊 11
2.2.3.3靜電吸引 13
2.2.3.4擴散 14
第三章 材料與方法 16
3.1實驗架構 16
3.2暴露系統建製 17
3.2.1小型暴露艙 19
3.2.2流量控制 21
3.3微粒暴露劑量評估與控制 22
3.3.1微粒沉積率 22
3.3.2暴露劑量控制 22
3.3.3氣動粒徑分析儀(APS) 22
3.3.4掃瞄式電移動度粒徑分析儀(SMPS) 22
3.3.5 DUST-TRAK 23
3.4細胞培養 23
3.5氣、液介面培養 Air–liquid interface 24
3.6細胞暴露實驗 24
3.7細胞計數及存活率分析 25
3.7.1 Trypan blue 染色法 25
3.7.2 MTT assay測試 25
3.8反應性氧化物種(ROS)量測 26
3.9統計分析 27
第四章結果與討論 28
4.1燃燒產生源粒徑分佈 28
4.1.1氣動粒徑分析儀 28
4.1.2掃瞄式電動度粒徑分析儀 29
4.2暴露系統穩定度 32
4.2.1燃燒產生源 32
4.2.2沉積率測試 35
4.3線香暴露對細胞的存活率分析 37
4.4線香暴露對細胞活性氧化物分析 42
第五章結論 46
參考文獻 47

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