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研究生:莊雅婷
研究生(外文):CHUANG, YA-TING
論文名稱:褐藻醣膠與UVC的結合處理可以無正常細胞毒性地優先驅動口腔癌細胞抗增生相關機制
論文名稱(外文):Combined UVC/fucoidan treatment preferentially drives antiproliferative mechanisms of oral cancer cells in non-normal cytotoxic manners
指導教授:張學偉
指導教授(外文):CHANG, HSUEH-WEI
口試委員:邱建智張嘉哲
口試委員(外文):CHIU, CHIEN-CHIHCHANG, CHIA-CHE
口試日期:2023-07-14
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:92
中文關鍵詞:多醣褐藻紫外線口腔癌優先抗增生活性氧凋亡
外文關鍵詞:polysaccharidebrown algalUVCoral cancerpreferential antiproliferationROSapoptosis
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褐藻醣膠(fucoidan; FN)是有高含量岩藻糖(fucose)的褐藻多醣體,據報導能抑制口腔癌細胞,但沒有正常細胞毒性。紫外線(ultraviolet C; UVC)已經應用在多種抗癌藥物的結合處理,具有協同抗癌作用。本論文探討UVC/FN結合處理在抗口腔癌研究的影響與機制。UVC/FN結合處理,對口腔癌細胞增生有協同抑制作用,但不會顯著影響正常口腔細胞生長。機制探討上,相對於單獨處理FN、UVC和正常細胞對照組而言,UVC/FN結合處理在口腔癌細胞,會優先誘發較高的細胞凋亡反應(subG1和annexin V的增加,以及多種caspases活化)、細胞活性氧化物、粒線體超氧化物和抗氧化物耗竭(glutathione; 穀胱甘肽)。與 FN或UVC單獨處理相比,FN/UVC在口腔癌細胞中會優先誘導氧化壓力,造成更多DNA斷裂和氧化性損傷。而抗氧化劑N-acetylcysteine會減少FN/UVC造成的這些氧化壓力反應機制。因此,本篇論文的貢獻是,提出新穎的FN/UVC結合處理方式,並證實褐藻醣膠FN有效地增加口腔癌細胞UVC敏感性,並且無副作用地抑制口腔癌細胞增生。
Fucoidan (FN), a brown algal-derived high-fucose polysaccharide, was reported to inhibit oral cancer cells under noncytotoxic drug safety to normal cells. Ultraviolet C (UVC) has been applied for synergistic anticancer effects by combining treatment with several anticancer agents. This thesis explores the impacts and mechanisms of UVC/FN on antioral cancer investigation. UVC/FN synergistically inhibited oral cancer cell proliferation without significantly harming normal oral cells. Mechanistically, UVC/FN-treated oral cancer cells preferentially triggered higher apoptosis responses (the increase of subG1 and annexin V, and caspase activations), cellular reactive oxygen species (ROS), mitochondrial ROS, and antioxidant depletion (glutathione) than FN or UVC and normal control cells. This preferential oxidative stress exerted by UVC/FN induced higher DNA breaks and oxidative DNA damage in oral cancer cells than in FN or UVC. These oxidative stress-responsive mechanisms of UVC/FN were alleviated by N-acetylcysteine. In conclusion, this thesis develops a novel combined treatment (UVC/FN) and validates that FN effectively improves UVC sensitization causing the antiproliferation of oral cancer cells without side effects.
中文摘要 3
英文摘要 4
1. 背景介紹 5
1.1. 口腔癌 5
1.2. 氧化壓力誘導細胞凋亡與DNA損傷 5
1.3. Fucoidan簡介 5
1.4. Fucoidan的功能與癌症相關研究 6
1.5. Ultraviolet C (UVC)與癌症相關研究 7
1.6. 結合處理的癌症治療應用 7
1.7. 假說 8
1.8. 研究目標 8
2. 實驗材料與方法 11
2.1. 細胞培養 11
2.2. 試劑配製 16
2.3. 細胞存活率(MTS assay)與協同作用分析(α值) 18
2.4. 細胞週期分析(Cell cycle analysis) 21
2.5. 細胞凋亡annexin V/7AAD分析(Annexin V/7AAD apoptosis analysis) 24
2.6. 細胞凋亡下游蛋白酶活性分析(Caspase 3 analysis) 27
2.7. 外源性細胞凋亡蛋白酶活性分析(Caspase 8 analysis) 30
2.8. 內源性細胞凋亡蛋白酶活性分析(Caspase 9 analysis) 33
2.9. 活性氧化物表現量測定(ROS assay) 36
2.10. 粒線體超氧化物表現量測定(MitoSOX assay) 39
2.11. Glutathione表現量測定(GSH assay) 42
2.12. DNA雙股斷裂測定(γH2AX) 44
2.13.氧化性DNA損傷分析(8-OHdG) 48
2.14. 統計方法 51
3. 結果 52
3.1. UVC/FN結合處理具協同抗口腔癌細胞增生 52
3.2. UVC/FN改變口腔癌細胞週期進行 52
3.3. UVC/FN引發口腔癌細胞凋亡 53
3.4. UVC/FN增加口腔癌細胞的下游細胞凋亡蛋白酶活性表現量 54
3.5. UVC/FN增加口腔癌細胞的外源性細胞凋亡蛋白酶活性表現量 55
3.6. UVC/FN增加口腔癌細胞的內源性細胞凋亡蛋白酶表現量 56
3.7. UVC/FN增加口腔癌細胞內活性氧化物 57
3.8. UVC/FN增加口腔癌細胞內粒線體超氧化物 58
3.9. UVC/FN增加口腔癌細胞內GSH表現量 59
3.10. UVC/FN引發口腔癌細胞DNA雙股斷裂 60
3.11. UVC/FN引發口腔癌細胞氧化性DNA損傷 61
4. 討論 62
4.1. FN與抗癌化療藥物的結合處理 62
4.2. UVC與抗癌化療藥物的結合處理 62
4.3. UVC/FN有選擇性殺死癌細胞的作用 63
4.4. UVC/FN誘發氧化壓力 63
4.5. UVC/FN誘導細胞凋亡與DNA損傷 64
4.6. 抗氧化劑NAC恢復UVC/FN誘導的抗增生、氧化壓力、細胞凋亡與DNA損傷 65
4.7. UVC的使用限制 66
5. 結論 67
6. 圖形與說明 69
7. 參考文獻 80
8. 發表文章與專利列表 87


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