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研究生:劉立敏
研究生(外文):Li-Min Liu
論文名稱:Econazole在人類口腔癌細胞誘導鈣離子湧出和細胞凋亡之研究
論文名稱(外文):Econazole-Induced Ca2+ Fluxes and Apoptosis in Human Oral Cancer Cells
指導教授:郭代璜郭代璜引用關係
指導教授(外文):Daih-Huang Kuo
學位類別:碩士
校院名稱:大仁科技大學
系所名稱:製藥科技研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:110
中文關鍵詞:鈣離子細胞凋亡econazole人類口腔癌細胞
外文關鍵詞:econazoleapoptosisCa2+human oral cancer cell
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本研究利用螢光染色Fura-2和WST-1實驗,探討Econazole如何影響人類口腔癌細胞(OC2) 中細胞內游離Ca2 +濃度 ([Ca2+]i) 和細胞的存活。Econazole在濃度依存性的實驗中發現,在給予1 M濃度及大於1 M濃度時,就能增加細胞內Ca2+的濃度。當去除細胞外的Ca2 +會降低部分鈣離子信號傳導。Aristolochic acid (phospholipase A2抑制劑) 和GF109203X (protein kinase C抑制劑) 具有抑制Econazole誘導的Ca2 +湧入的作用。在無鈣離子的培養液,人類口腔癌細胞(OC2)先以1 M thapsigargin (內質網Ca2 +幫浦抑制劑) 處理過後,30 M econazole未能引起細胞內Ca2 +濃度的增加。用2 M U73122來抑制phospholipase C 卻能顯著抑制econazole誘導細胞內Ca2 +濃度的上升。在濃度5至70 M之間,Econazole會隨著濃度的增加使細胞存活率下降,而具有細胞毒性的作用。50 M Econazole所引起的細胞毒性作用,不會因為加入了BAPTA/AM (Ca2 + 螯合劑)螯合細胞內Ca2+後降低其毒性,反而加劇了細胞毒性。ERK/MAPK訊號傳導抑制劑PD98059 (10 M) 會增強20 M econazole所誘導的細胞死亡。從PI staining實驗中可得知,econazole誘導細胞凋亡的在濃度10-70M 之間。總結來說,在人類口腔癌細胞 (OC2) 中,econazole誘導的細胞內Ca2 +濃度的升高,是因為Ca2 +從內質網釋放和Ca2 +從phospholipase A2/ protein kinase C調節Ca2 +通道中湧入所造成的。除此之外,econazole所造成細胞凋亡,可能是與Ca2 +和ERK/MAPK訊號傳導路徑的調控有相關性。
The effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations of and above 1 M increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole-induced Ca2+ influx was sensitive to blockade of aristolochic acid (a phospholipase A2 inhibitor) and GF109203X (a protein kinase C inhibitor). In Ca2+-free medium, after treatment with 1 M thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30 M econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2 M U73122 substantially suppressed econazole-induced [Ca2+]i rise. At concentrations between 5 and 70 M econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 M econazole was not enhanced by prechelating cytosolic Ca2+ with BAPTA/AM. The ERK/MAPK inhibitor PD98059 (10 M) also enhanced 20 M econazole-induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10-70 M. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/protein kinase C-regulated Ca2+ channels. Furthermore, econazole caused apoptosis that was regulated by Ca2+ and ERK/MAPK.
誌謝 I
中文摘要 III
Abstract IV
目錄 V
表目錄 VIII
圖目錄 IX
第壹章 緒論 1
第一節 口腔癌 1
(一) 什麼是口腔癌? 1
(二) 口腔癌的流行病學 1
(三) 口腔癌的成因 2
(四) 人類口腔鱗狀細胞癌的臨床病徵 5
(五) 人類口腔鱗狀細胞癌的病理分期 6
(六) 人類口腔鱗狀細胞癌的治療 6
第二節 細胞週期 8
第三節 細胞週期調控相關因子 12
(一) Cyclins與Cyclin-dependent kinases (Cdks) 12
(二) Cyclin-dependent kinases inhibitors(CdkIs) 13
(三) G1/S Transition 15
(四) S phase progression 15
(五) G2/M transition 16
(六) MAP Kinase 17
第四節 細胞死亡 18
(一) 細胞壞死 26
(二) 細胞凋亡 27
第五節 細胞凋亡訊息傳導途徑 28
(一) 外在路徑Extrinsic Pathway 28
(二) 內在路徑Intrinsic pathway 33
(三) 鈣離子 40
(四) 活性氧化物 43
第六節 Econazole 44
第貳章 研究目的 47
第參章 材料與方法 48
第一節 實驗儀器與材料 48
(一) 實驗儀器 48
(二) 實驗藥品 48
(三) 實驗溶液 48
第二節 實驗方法 49
(一) 細胞培養Cell culture 49
(二) [Ca2+]i測量 49
(三) 細胞活性試驗Cell viability assays 50
(四) Flow cytometry檢測細胞凋亡 51
(五) 統計分析 51
第肆章 結果 52
(一) Econazole 對於 [Ca2+]i 的影響 52
(二) Econazole對Mn2+ 細胞內轉移的作用 56
(三) Aristolochic acid和GF 109203X 影響Econazole誘導的[Ca2+]i上升 58
(四) 細胞內Ca2+鈣蓄池和Econazole誘導的[Ca2+]i 上升 60
(五) Phospholipase C參與在Econazole誘導[Ca2+]i的上升 60
(六) Econazole對細胞存活的影響 61
(七) Econazole誘導[Ca2+]i的升高和細胞死亡的關係 61
(八) 在Econazole誘導的細胞死亡作用中MAPK/ERK所扮演的角色 66
(九) Econazole誘導的細胞死亡可能有細胞凋亡的參與 66
第伍章 討論 69
第陸章 參考文獻 71
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