探討氧化緊迫與trans,trans-2,4-decadienal促進人類氣管上皮細胞生長及細胞激素表現等作用的相關性

Investigating the role of oxidative stress in trans,trans-2,4-decadienal induced cell proliferation and cytokines expression in human bronchial epithelial cells

盧慧詩(Wai-Sze Lo)

指導教授 ： 林嬪嬪

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2005年)

摘要

目前肺癌高居台灣地區男性及女性癌症死因第二及第一位。許多研究顯示肺癌的發生與暴露環境中的致癌物或致突變物有關。流行病學研究結果顯示，台灣女性肺癌的發生可能與經常暴露烹飪油煙有關。已知烹飪油經加熱後會產生多種醛類，而烹飪油中的多不飽合脂肪酸經過氧化，其中以trans, trans-2,4-decadienal (tt-DDE or 2,4-De)的致突變性最高，在肺癌細胞造成氧化性的傷害，為了提供更多科學證據以了解tt-DDE的暴露是否與女性肺癌的發生有關，本論文目的是探討tt-DDE在人類支氣管上皮細胞(BEAS-2B)的生物作用，並探討抗氧化劑-維生素C (vitamin C)和N-acetylcysteine (NAC)是否能降低tt-DDE所誘發的生物效應。我們利用MTT assay分析tt-DDE對細胞生長速度之影響，用流式細胞儀分析細胞內氧化緊迫之情形，並利用the real-time RT-PCR相對定量特定基因的mRNA。首先我們發現急性處理0.1 或 1 micromole tt-DDE 48小時或29天後，增加細胞內氧化緊迫，而5 micromole tt-DDE會選擇性地殺死非肺癌的細胞; 此外，經0.1 micromole 或1 micromole tt-DDE處理 48小時或32天後，還原態與氧化態glutathione的比例(GSH/GSSG)和glutathione (GSH)的百分比的濃度，顯著較DMSO控制組減少，而glutathione (包括氧化態及還原態glutathione)的總量和氧化態glutathione (GSSG)濃度卻增加。因此推論tt-DDE增加細胞氧化性緊迫，促使GSH氧化成GSSG，不過也增加細胞內glutathione總量。我們以非致死劑量的tt-DDE (0.1 或 1 micromole) 處理BEAS-2B細胞30-45天後，反而增加細胞生長速度，且具有劑量反應關係，其中經1 micromole tt-DDE處理45天後，增加TNF-alpha 和 IL-1beta mRNA量及蛋白活性，同時也增加IL-6 mRNA量。此外，共同處理抗氧化劑(vitamin C or N-acetylcysteine)與tt-DDE後，能減少因tt-DDE增加細胞生長速度及細胞激素mRNA量等作用。TNF-alpha、IL-1beta 及IL-6是發炎前期所分泌的細胞激素，許多腫瘤促進因子均會增加其分泌，也有實驗結果證實TNF-alpha、 IL-1beta 及IL-6具有促進腫癌的作用。另一方面，也檢測tt-DDE對調控細胞週期的p21家族，例如p21、p27和p57 mRNA的影響，發現經1 micromole tt-DDE處理45天後，只會顯著降低p27 mRNA累積量，但不影響p21及p57 mRNA累積量。因此推測tt-DDE是經由增加細胞內氧化緊迫、促進細胞激素基因和蛋白表現及降低p27 mRNA量，進而促進細胞生長，可能具有腫瘤促進的作用。

關鍵字

細胞激素

並列摘要

Lung cancer is the first and the second leading cause of cancer death among females and males in Taiwan, respectively. Several studies showed that lung cancer is associated with exposure to the environmental carcinogens and mutagens. Epidemiological studies suggested that exposure to cooking oil fumes (COF) increased the risk of female lung cancer in Taiwan. Dienaldehydes are by-products of peroxidation of polyunsaturated lipids and found in many foods and food-products. Trans, trans-2,4-decadienal (tt-DDE or 2,4-De) is a type of dienaldehydes and is abundantly found in heated oils, including COF. The aim of this study was to investigate the biological effects induced by tt-DDE and the possible protective mechanism of antioxidant-vitamin C and N-acetylcysteine (NAC) in immortalized human bronchial epithelial cells BEAS-2B. Cell proliferation was assayed using MTT method. Intracellular reactive oxygen species (ROS) was analyzed by flow cytometry and the genes expression was determined with the real-time RT-PCR method. We found that treatment with 0.1 micromole or 1 micromole tt-DDE for 48 hr or 29 days significantly increased intracellular oxidative stress in lung cells. Furthermore, 5 micromole tt-DDE selectively reduced viable cell numbers in BEAS-2B cells, but not in lung cancer cells. On the other hands, treatment with 0.1 micromole or 1 micromole tt-DDE for 48 hr or 32 days decreased the GSH/GSSG ratio and GSH (reduced form), but increased total GSH (including reduced and oxidized forms) and GSSG (oxidized form). Therefore, these data suggested that tt-DDE induced intracellular oxidative stress might urge the GSH (reduced form) to turn into GSSG, and also increase the total glutathione. Long-term exposure (30-45 days) with sub-lethal doses (0.1 or 1 micromole) of tt-DDE significantly increased cell growth of BEAS-2B cells. On day 45, TNF-alpha and IL-1 beta gene expression and secretion was significantly increased in 1 micromole tt-DDE-treated cells, and also IL-6 mRNA levels were increased. In contrast, co-treatment with vitamin C or N-acetylcysteine, antioxidants, partially pervented tt-DDE induced cell proliferation, decreased pro-inflammatory genes expression. TNF-alpha、IL-1beta and IL-6 are pro-inflammatory cytokines. Several tumor promoters were shown to increase their secretion. On the other hands, we found that p27 mRNA levels reduced by 1 micromole DDE- treated for 45 days in BEAS-2B cells, and this reduction was protected by NAC co-treatment. It has been reported that TNF-alpha and IL-1beta had tumor promotion function, and many tumor promoters increased ROS generation. Therefore, tt-DDE might be a tumor promoter via increasing intracellular ROS in human bronchial epithelium cells.

並列關鍵字

cytokine

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