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研究生:楊慧婷
研究生(外文):Huei-Ting Yang
論文名稱:彗星分析法評估氯乙烯聚合工人DNA傷害之研究
論文名稱(外文):Dection of DNA damage with comet assay in polyvinyl chloride workers
指導教授:鄭尊仁鄭尊仁引用關係
指導教授(外文):Tsun-Jen cheng
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:74
中文關鍵詞:聚氯乙烯彗星分析DNA單鼓斷裂
外文關鍵詞:polyvinyl chloridecomet assayDNA single-strand break
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中文摘要
氯乙烯是人類致癌物質,許多流行病學研究都指出氯乙烯暴露和肝癌之間的相關性。DNA產生傷害是癌症發生一個很關鍵性的步驟,所以過去有許多氯乙烯暴露和DNA傷害的研究,除了形成DNA鍵結物外,細胞遺傳學方面也發現氯乙烯會造成染色體變異、姊妹染色體交換、微核和DNA股斷裂的產生,不過低濃度氯乙烯暴露 (<5ppm)和DNA傷害的相關性並不清楚,因此我們以一種新的分析方法即”彗星分析法”評估聚氯乙烯工人周邊淋巴球DNA單股斷裂的情形,再以GC-MS評估工作日隔天早晨尿中TdGA (Thiodiglycolic acid)的濃度,過去的研究已經指出空氣中氯乙烯的濃度和尿中代謝產物TdGA之間具有顯著相關,也就是說尿中TdGA是評估氯乙烯暴露較好的內在劑量指標,最後再分析尿中代謝產物TdGA與DNA單股斷裂的相關性。
本研究為三家氯乙烯聚合工廠的男性作業員工,共26人,除了採集氯乙烯暴露當天下午的血液和氯乙烯暴露隔天早晨的尿液樣本外,所有工人皆以問卷訪視的資料獲得氯乙烯聚合工人的個人基本資料、生活習慣、疾病史及工作概況等。
研究結果顯示氯乙烯尿中代謝產物TdGA的濃度與彗星分析評估DNA單股斷裂的發生具有顯著相關(P<0.01),而且尿中TdGA濃度>1.5 mg/g Cr (氯乙烯個人採樣濃度>1 ppm)的研究對象其彗星分析結果% of tail length、 % of tail intensity和Tailmoment也顯著高於尿中TdGA濃度≦1.5 mg/g Cr (氯乙烯個人採樣濃度≦1 ppm)的研究對象(P<0.05)。而抽菸、年齡和飲食習慣等潛在干擾因子在本研究對彗星分析的結果沒有產生影響。
總而言之。我們的研究結果顯示在低濃度的氯乙烯暴露下(0.05-13.38 ppm),仍然可以觀察到在環境氯乙烯濃度大於1ppm的工人相較於小於1ppm的有比較多的DNA單股斷裂(P<0.05),另外氯乙烯暴露濃度1-5ppm的工人其彗星分析結果亦高於氯乙烯暴露濃度<1ppm,但沒有達到統計上的顯著差異。雖然本研究不能證實彗星分析相較於其他遺傳學分析法有比較高的偵測敏感性,但是卻指出即使在符合法規的情況下,即氯乙烯八小時時量平均濃度≦TWA(5ppm),仍可能有其危害性存在。

Abstract
Vinyl chloride monomer (VCM) has been classified as a human carcinogen. A large number of epidemiology studies have demonstrated an association between VCM exposure and liver cancer. DNA damage is a critical step in the carcinogenesis. DNA adducts and cytogenetic methods including chromosomal aberrations, sister chromatid exchange frequency and micronuclei have been used to assess DNA damage in VCM exposure workers. However, the dose-response relationship between the low dose exposure to VCM and DNA damage remains unclear.
A total of 33 male workers in three polyvinyl chloride factories were included in our study. Blood samples at the end of the working shift and urine samples at the commencement of the following shift were collected. Questionnaires were administered to obtain demographic data, detailed history of occupation, and lifestyles. We used comet assay to assess the DNA single strand-strand breaks (SSB) in peripheral lymphocyte. Their urinary Thiodiglycolic acid (TdGA) concentrations were also determined using GC-MS. Our previous study have demonstrated a significant dose-response relationship between the concentration of VCM in the air and urinary TdGA. In this study, we will investigate the relationship between TdGA and DNA SSB.
Our results revealed a significant relationship between the urinary TdGA levels and DNA SSB (P<0.01). The % of tail length and % of tail intensity were also significantly increased in workers had higher TdGA concentrations (TdGA>1.5 mg/g Cr corresponding to air VCM exposure levels than 1 ppm) comparing to those had low TdGA concentrations (TdGA<1.5 mg/g Cr corresponding to air VCM levels less than 1 ppm) (P<0.05). No significant difference between TdGA and potential confounders including cigarette smoke, age and lifestyles were observed in our study.
In conclusions, there is no evidence suggesting that comet assay is more sensitivity than standard cytogenetic methods in detecting VCM-induced DNA damage. However, our results revealed that VCM may cause DNA damage at air levels below current TWA-PEL (5ppm).

摘 要 2
ABSTRACT 4
圖表目錄 8
第一章 前言 10
第二章 文獻回顧 11
2.1 氯乙烯與癌症的流行病學研究 11
2.2 氯乙烯暴露和DNA傷害之研究 12
2.2.1 DNA 鍵結物 (生物有效劑量指標) 12
2.2.2 細胞遺傳學 (早期生物效應指標) 13
(1) 染色體變異 13
(2) 姊妹染色體交換和微核 14
(3) 單股染色體斷裂 14
2.3 彗星分析法 15
2.3.1 彗星分析的原理及優缺點 15
2.3.2 彗星分析在環境職業醫學的應用 17
2.3.3 氯乙烯與SSB之相關 18
2.3.4 影響彗星分析DNA傷害的因素 18
(1) 抽菸 18
(2) 年齡與性別 19
(3) 飲食習慣 19
(5) DNA股內交錯連結 20
(6) 細胞凋亡現象 20
第三章 材料與方法 21
3.1 研究對象: 21
3.1.1 流行病學資料 21
3.1.2 氯乙烯暴露分組 21
(1) 環境採樣 21
(2) 個人採樣 21
(3) 尿中Thiodiglycolic acid的濃度 22
3.2 GC-MS分析尿液TdGA 22
3.2.1 尿液收集 22
3.2.2 校正曲線製作 22
3.2.2 尿液前處理與化學物衍生 23
(1) 尿中肌肝酸的測定 23
(2) 萃取 23
(3) 化學衍生 23
(4) 方法偵測極限 23
3.2.3 儀器 23
(1) 氣相層析儀 23
(2) 質譜儀 24
3.3 彗星分析評估氯乙烯聚合工人DNA傷害情形 24
3.3.1 淋巴球的收集 24
3.3.2 彗星分析實驗原理 24
(1) 玻片的製作 (膠的製作) 24
(2) 浸泡鹼性的lysis buffer 25
(3) 浸泡電泳液 25
(4) 電泳 25
3.3.3 彗星分析實驗步驟 25
3.3.4 影像分析 27
(1) 攝影及存檔 27
(2) 軟體分析 27
3.3.5 統計分析 28
第四章 結果 29
第五章 討論 32
第六章 參考文獻 37

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