博碩士論文 110821015 詳細資訊




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姓名 林晏如(Yen-Ju Lin)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 PM2.5 暴露對人類結腸腺癌 Caco-2 細胞的不良影響 -氧化壓力、發炎、細胞增殖及自噬損傷
(Adverse effects of PM2.5 exposure in human colonic adenocarcinoma Caco-2 cells-oxidative stress, inflammation, cell proliferation, and autophagy flux impairment)
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摘要(中) Particulate matter (PM) 2.5是一種直徑小於或等於2.5直徑的懸浮微粒。大部分來源來自人為排放,其中包括工業燃燒和汽機車排放的廢氣。目前,PM 2.5已被國際癌症研究機構(International Agency for Research on Cancer, IARC)認定為一級致癌物,可通過呼吸系統進入人體,造成肺部損害,引發呼吸系統疾病,甚至進一步引發心血管疾病。此外,長期接觸 PM 2.5與結直腸癌(colorectal cancer, CRC)的癌症進展和死亡率相關。多項研究報告稱,PM 2.5改變了腸道微生物菌群並使其失衡,可能進一步導致代謝疾病和炎症,然而,分子信號傳導機制仍不清楚。本研究中,使用購自美國的標準品SRM (standard reference material of fine particulate matter) 2786在不同的暴露濃度與時間下,探討SRM 2786對Caco-2結腸腺癌的不良反應。我們發現在SRM 2786 處理的Caco-2中,能引起細胞增殖、活性氧(Reactive oxygen species, ROS)產生、環氧合酶 (Cyclooxygenase-2, COX-2)、粘著斑激酶 (Focal adhesion kinase, FAK)磷酸化、NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells)途徑中p65磷酸化及IL-1 beta (Interlukin-1 beta)表現量顯著增加。而抗氧化劑 (Glutathione, GSH)可顯著降低暴露於 SRM 2786的Caco-2細胞中ROS的產生和細胞增殖, NADPH oxidase 1 (NOX-1)及NADPH oxidase 4 (NOX-4)抑制劑也可顯著減少ROS的產生,SRM 2786還會增加了血紅素加氧酶 1 (Heme oxygenase-1, HO-1)和NAD(P)H-醌氧化還原酶 1 (NQO1)抗氧化酶的表現量,另外,以COX-2及FAK的抑制劑可顯著降低細胞增殖。同時,SRM 2786增加了LC3II和p62的表現量並引發溶酶體膜完整性的喪失,這些結果顯示 SRM 2786損害了自噬活化和溶酶體功能,損害細胞自噬流(autophagic flux)。暴露SRM 2786亦會導致Occludin及TJP2 (Tight junction protein 2)增加而ZO-1 (zonula occludens-1)則是減少,代表有可能損傷腸道基本屏障的功能,因此,SRM 2786對大腸癌細胞造成氧化壓力、發炎反應、細胞增殖和自噬損傷等不良反應,有可能會增加大腸癌的風險。
摘要(英) Particulate matter (PM) 2.5 is a fine particulate matter with a diameter less than or equal to 2.5 μm. Common sources come from anthropogenic emissions, which include industrial combustion and emissions from automobiles and locomotives. At present, PM2.5 has been identified as a primary carcinogen by the International Agency for Research on Cancer (IARC), which can enter the human body through the respiratory system, causing lung damage, respiratory diseases, and even further cardiovascular diseases. Furthermore, long-term exposure to PM2.5 is associated with cancer progression and mortality in colorectal cancer (CRC). Several studies have reported that PM alters and unbalances the gut microbiota, which may further contribute to metabolic disease and inflammation. However, the molecular signaling mechanism remains unclear. In this study, the standard reference material of fine particulate matter (SRM) 2786 purchased from the United States was used to investigate the adverse effects of SRM 2786 on Caco-2 colon adenocarcinoma under different exposure concentrations and time. We found that in SRM 2786-treated Caco-2, cell proliferation, Reactive oxygen species (ROS) production, cyclooxygenase (COX-2), Focal adhesion kinase (FAK) phosphorylation, p65 phosphorylation in NF-kB (Nuclear Factor kappa-light-chain-enhancer of activated B cells) pathway, and IL-1 beta (Interlukin-1 beta) levels increased significantly. Antioxidant GSH (Glutathione) can significantly reduce ROS production and cell proliferation in Caco-2 cells exposed to SRM 2786. NADPH oxidase 1 (NOX-1) and NADPH oxidase 4 (NOX-4) inhibitors can also significantly reduce ROS levels in Caco-2 cells after SRM 2786 exposures. PM also increased the levels of Heme oxygenase 1 (HO-1) and NAD(P)H-quinone oxidoreductase 1 (NQO1) antioxidant enzymes. In addition, inhibitors of COX-2 and FAK can significantly reduce cell proliferation. Meanwhile, SRM 2786 increased the levels of LC3II and p62 and triggered the loss of lysosomal membrane integrity, suggesting that SRM 2786 impaired autophagic flux and lysosomal function. Exposure to SRM 2786 also resulted in an increase in Occludin and TJP2 (Tight junction protein 2) and a decrease in ZO-1 (zonula occludens-1), which may impair the function of the basic intestinal barrier Our results suggest that adverse effects of SRM 2786 exposure on CRC cells include oxidative stress, inflammation, cell proliferation, and autophagy impairment, which may increase the risk of CRC.
關鍵字(中) ★ 細胞自噬
★ 大腸癌
★ 氧化壓力
關鍵字(英) ★ PM2.5
論文目次 中文摘要 I
Abstract II
致謝 III
目錄 IV
圖目錄 VI
縮寫檢索表 VII
第一章 緒論 1
1.1懸浮微粒 (Particulate Matter) 1
1.2 SRM 2786 (Standard reference material of fine particulate matter 2786) 2
1.3活性氧與抗氧化路徑ROS (Reactive oxygen species) and Nrf2(Nuclear factor erythroid 2-related factor 2) pathway 3
1.4細胞自噬 (Autophagy) 4
1.5緊密連接 (Tight junction) 5
1.6 NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) 6
第二章 研究動機 8
第三章 實驗材料與方法 8
3.1實驗材料 9
3.1.1實驗細胞株 9
3.1.2實驗材料 9
3.1.3 試劑 9
3.1.4 耗材 10
3.1.5 儀器 11
3.2實驗方法 11
3.2.1 Caco-2培養方式 11
3.2.2 SRM 2786製備方式 12
3.2.3 BCA蛋白質濃度測定 ( Pierce™ BCA Protein Assay ) 12
3.2.4西方墨點法 ( Western Blot ) 13
3.2.5細胞增殖檢測 (Cell Counting Kit-8, CCK-8 assay) 14
3.2.6活性氧檢測 (2′,7′-Dichlorodihydrofluorescein diacetate, DCF-DA assay) 15
2.2.7免疫螢光染色及共同免疫螢光染色 (Immunofluorescence Staining and Co-immunofluorescent staining) 15
3.2.8活染溶酶體 (BioTracker 560 Orange Lysosome Dye) 16
3.2.9 細胞存活率試驗(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT Assay) 16
第四章 結果 17
4.1 Caco-2細胞在SRM 2786的暴露下引起細胞增生 17
4.2 Caco-2細胞在SRM 2786的暴露下引發氧化壓力和抗氧化機制 17
4.3 Caco-2在SRM 2786暴露下引發COX-2訊號活化 18
4.4 Caco-2細胞在SRM 2786的暴露下引起NF-κB活化 18
4.5 Caco-2在SRM 2786暴露下引發IL-1 Beta表現增加 19
4.6 Caco-2細胞在SRM 2786的暴露下引發細胞自噬反應失調 19
4.8 Caco-2細胞在SRM 2786的暴露下引起 FAK (Focal Adhesion Kinase)異常表現 21
4.9 Caco-2細胞在SRM 2786的暴露下引起 tight junction異常表現 22
第五章 討論 23
第六章 參考文獻 28
圖 37
附錄一 55
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指導教授 羅月霞(Yueh-Hsia Luo) 審核日期 2023-7-20
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