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)

指導教授 ： 吳佩芝

長榮大學/健康科學學院/職業安全與衛生學系(所)/碩士(2013年)

https://doi.org/10.6833/CJCU.2013.00234

摘要

II

中文摘要
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，會產生甲醛二次有機氣膠。
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，並使
用 MTT assay 及 Trypan Blue 來探討細胞的存活率、使用 2’7’-Dichlorofluorescein
diacetate (DCFH-DA)及螢光酵素免疫分析儀量測 ROS 含量，並使用聚合酶鏈反
應( polymerase chain reaction, PCR)來量測細胞在暴露後發炎指標(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。
以 Trypan Blue 來判斷細胞的存活率，結果顯示控制組的細胞存活率可以達
到 72％-85%，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％。單純暴露不同臭氧濃度， ROS 產量會隨著暴露的時間而增加，在暴露
臭氧混合 β-蒎烯 2 小時後，ROS 的產生量明顯的較單純暴露臭氧來的高，也可
以看出 ROS 的產生量與暴露的濃度呈現明顯的劑量效應關係。在發炎指標方
面，暴露不同濃度的臭氧後，介白素 8 的釋放量呈現劑量效應關係，以暴露 2
小時不同濃度臭氧的劑量效應關係最為明顯。而細胞在暴露 2 小時 30 ppb 臭氧
混合 β-蒎烯，介白素 6 有呈現劑量效應關係，但是在暴露 4 小時、6 小時 30 ppb
及 60 ppb 上，劑量關係則變的不明顯。在暴露不同時間及濃度的臭氧及臭氧混
合精油後，又以介白素 8 劑量效應關係最為明顯，也最穩定。
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
III

白素 8 而言，介白素 8 的釋放量較為明顯也較穩定。

關鍵字

臭氧 ； β-蒎烯 ； 甲醛 ； 介白素 8 ； 介白素 6 ； 氣 液介面直接暴露系統

並列摘要

IV

Abstract
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 triacetate (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%. 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 mixtures. 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
V

formaldehyde and ofer derivatives from the mixtures of ozone andβ-pinene which
is one of the common sources of nature fragrance in indoor environment.

並列關鍵字

ozone ； β-pinene ； IL-8 ； IL-6 ； Air-Liquid Interface (ALI)

參考文獻

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