利用氣、液介面細胞株直接暴露系統探討臭氧及β-蒎烯氧化產物對細胞發炎反應之影響

Exploring the Impact of Ozone and β-pinene Oxidative Products on Cell Inflammation by Using Air-Liquid Interface Expose System

梁景惠(Ching-Hui Liang) ； 吳佩芝(Pei-Chih Wu) ； 戴聿彤(Yu-Tung Dai)

《勞動及職業安全衛生研究季刊》 23卷3期 (2015/09) Pp. 322-334

摘要

臭氧是一種擁有強氧化力的氣體，許多研究證實吸入臭氧會引發各種健康問題，包括胸痛、咳嗽、喉嚨發炎，引發支氣管炎、肺氣腫、哮喘惡化等，臭氧的高反應性也會造成氧化壓力、增加細胞膜的通透性、造成細胞間反應性氧化物種（Reactive oxygen species, ROS）的增加甚至會造成細胞的損傷，在許多研究中也已經證實，臭氧與β-蒎烯混合後，會產生甲醛及二次有機氣膠。目的：本研究以一氣液界面直接暴露系統，讓A549細胞暴露不同濃度（30、60、120 ppb）和不同時間（2、4、6小時）的臭氧和臭氧混合帖烯類物質-β-蒎烯，細胞在暴露後量測發炎指標（介白素6、介白素8）的產生量，並且探討是否與暴露時間及濃度呈現劑量效應關係。研究方法：使用MTT assay及Trypan Blue來探討細胞的存活率並使用2'7'-Dichlorofluorescein diacetate（DCFH-DA）、螢光酵素免疫分析儀量測ROS含量及聚合酶鏈反應（polymerase chain reaction,PCR）量測發炎前驅物質IL-6和IL-8的生成量。結果：結果顯示控制組的細胞存活率可以達到74%，隨著暴露時間的增加及臭氧濃度的增加，存活率很明顯的減少至52％。MTT的實驗結果也可以看出細胞活性隨著暴露時間及暴露劑量的增加，吸光值有明顯的降低，而統計結果也顯示出細胞的活性隨著不同的暴露濃度、劑量有顯著差異；在暴露不同臭氧濃度後，細胞 ROS產量會隨著暴露劑量的增加，ROS產生量明顯比單純暴露臭氧來的高，也可以看出ROS的產生量與暴露的劑量呈現明顯的劑量效應關係。發炎指標方面，暴露不同濃度的臭氧後，介白素6需在高濃度臭氧（120ppb）才呈現出劑量效應關係；而介白素8的釋放量呈現明顯劑量效應關係。本研究結果顯示出，細胞在暴露到二次氣膠以及臭氧混合β-蒎烯主要衍生物的毒性較單純暴露臭氧來的高（細胞活性、ROS、IL-6、IL-8）。從細胞暴露後發炎指標的測定中發現，2小時及4小時是較好的暴露時間，就介白素6及介白素8而言，介白素8的釋放量較明顯也較穩定。

關鍵字

臭氧 ； β-蒎烯 ； 甲醛 ； 介白素8 ； 介白素6 ； 劑量效應關係

並列摘要

Ozone is a strong oxidant gas. Many studies have showned that ozone can cause a variety of health problems including chest pain, coughing, throat irritation, bronchitis, emphysema, and worsen asthma. The high reactivity of ozone also cause oxidative stress to increase the permeability of the cell membrane, and reactive oxygen species (ROS) that cause cell damage, some studies have also confirmed that the ozone mixed with β-pinene, will produce secondary aerosols and formaldehyde. Our study use air-liquid interface with A549 cells to expose to different concentrations (30,60,120 ppb) at different times (2,4, 6 hours) and the mixture with ozone and β-pinene. We use Trypan Blue to analyze cell viability and use 2'7'-Dichlorofluorescein diacetate (DCFH-DA) with fluorescence enzyme immunoassay analyzer to measure the release of ROS from cells. Polymerase chain reaction (PCR) was to measure the production of inflammatory markers (IL-6, IL-8) after exposure scenario different. The cell viability determined by trypan blue, in the control group were 72%-85%, with the increase of exposure time and ozone concentration, the viability reduced to 52%. From the experimental results of MTT cell activity can also be seen with increasing exposure time and the exposure dose, the absorbance values are significantly lower, the results of statistical also showed cell activity are significant differences in the dose with different exposure concentrations. ROS production in different ozone exposures has shown a dose dependent effects. The production of ROS were significantly increase when expose to the mixtures of ozone and β-pinene at 2 hours. IL-6 and IL-8 also show a dose-dependent effect with increasing ozone and ozone-β-pinene mixures. Our study demonstrate exposure times, levels of ozone and ozone-β-pinene mixture, would trigger a dose-dependant relationships in viability, ROS, IL-6, IL-8. The results of this study also highlight the importance of secondary aerosol and formaldehyde and ofer derivatives from the mixtures of ozone and β-pinene which is one of the common sources of nature fragrance in indoor enviroment.

並列關鍵字

Ozone ； β-pinene ； IL-8 ； IL-6 ； Formaldehyde ； Dose-Dependant relationships

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