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研究生:林軍伯
研究生(外文):Jyun-Bo Lin
論文名稱:生育醇對人類癌細胞鈣離子衡定及細胞毒性之影響
論文名稱(外文):The calcium homeostasis and cytotoxicity effect ofgamma-tocopherol on human cancer cells
指導教授:劉佩珊劉佩珊引用關係
指導教授(外文):Pei-Shan Liu
學位類別:碩士
校院名稱:東吳大學
系所名稱:微生物學系
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:66
中文關鍵詞:生育醇鈣離子牛血清蛋白蕈毒鹼受器細胞凋亡
外文關鍵詞:tocopherolcalciumbovine serum albuminmuscarcinic receptorapoptosis
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富含維他命E之食物具有益處已被廣為人知,維他命之功能可以清除自由基並防止個體內DNA被傷害。而維他命E指的是一群結構相近的化合物,依其結構差異性而有不同性質。gamma-tocopherol,是食品中含量最豐富的維他命E,因此本篇研究以gamma-tocopherol為材料,以釐清其在鈣離子訊號、細胞生長和死亡上的角色。使用細胞為人類表皮HeLa細胞,人類胃癌AGS細胞,和人類神經纖維母細胞SK-N-SH。以Fura-2鈣離子測定法進行細胞內鈣子濃度偵測,以及西方點漬法偵測訊號路徑內蛋白質含量。細胞的增生和凋亡,則使用MTT、subG1,粒腺體膜電位,DNA片段化和caspase表現程度來判定。我們首度發現gamma-tocopherol可以誘導細胞內鈣濃度的上升,並在多種細胞上可抑制鈣離子管道,包括尼古丁受器,P2X受器、膜電位離子管道、和鈣離子通透劑A23187及ionomycin等。我們也發現 gamma-tocopherol和牛血清蛋白 (Bovine serum albumin; BSA)的混合物,能夠導致HeLa細胞和AGS細胞的死亡,但SK-N-SH則否。相似的特點也也在人類血清蛋白 (Human serum albumin; HAS) 觀察到。gamma-tocopherol和BSA混合物可導致SubG1的增加,DNA片段化,Caspase3和9的表現,8則無。Caspase3,9之抑制劑可以有效拮抗BSA/ gamma-tocopherol毒性,但caspase8抑制劑則否。在不同的細胞,觀察到BSA/ gamma-tocopherol造成不同的死亡的程度,推測可能是在不同細胞上有不同分子機轉。本篇論文是首度發現gamma-tocopherol可以干擾鈣的平衡,且BSA/ gamma-tocopherol擁有造成細胞凋亡的能力。
Vitamin E rich diets benefits are well established, to neutralize free radicals and prevent DNA damage in the body. The term vitamin E represents several structurally related compounds, each differing in their potency and molecular mechanisms. Gamma-tocopherol, the most abundant one in the food, is investigated in this study to clear its roles on calcium signaling, cell growth and death in cells, including human epithelial carcinoma (HeLa) cells, human gastric cancer (AGS) cells, and human neuroblastoma (SK-N-SH) cells. Fura-2 measurements were used for [Ca2+]i measurement, and Western Blotting for detection the level of protein in the signaling pathway. Cell proliferation and apoptosis were measured using the MTT assay, sub-G1 phase, mitochondria membrane potential, DNA laddering and caspase activation. We found a novel character of gamma-tocopherol as inducing a cytosolic calcium ([Ca2+]i) rise and blocking calcium channel in various types, including nicotinic receptors, P2X receptors, voltage-gated ion channels and calcium ionophore A23187 and ionomycin. We also found the mixture of gamma-tocopherol and bovine serum albumin will cause death of HeLa and AGS cells but not SK-N-SH cells. Similar character also observed in human serum albumin. Bovine serum albumin/gamma-tocopherol treatment resulted in increase of subG1, DNA laddering, caspase 3, and 9, but not caspase 8. The inhibitors of caspase 3, 9 but not 8 were effective to block the cytotoxicity of Bovine serum albumin/gamma-tocopherol. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of Bovine serum albumin/gamma-tocopherol to induce cell death. This is the first study to demonstrate that gamma-tocopherol has some interference on [Ca2+]i and Bovine serum albumin/gamma-tocopherol has cytotoxicity to cause apoptosis.
縮 寫 表 III
中文摘要 V
Abstract VI
前 言 1
文獻回顧 3
藥 品 8
實驗方法 11
一、細胞培養 10
二、細胞色素排除法-Trypan Blue (Eibl et al., 2003) 10
三、MTT Kit分析細胞存活 (Zaslau et al., 2004) 11
四、鈣訊之偵測 (Liu et al., 1996) 11
五、細胞週期(cell cycle)與DNA指數分析 (Yao et al., 2006) 12
六、粒腺體膜電位變化偵測 (Yao et al., 2006) 12
七、caspase3、8、9偵測 (Yao et al., 2006) 13
八、細胞全蛋白的製備 (Pandey, 2006) 13
九、蛋白質濃度的測定 13
十、西方墨點法 (Brandstrom et al., 2001) 14
結 果 17
一、gamma-tocopherol引起細胞內鈣離子濃度的上升 15
二、gamma-tocopherol抑制多種受器引起之[Ca2+]i上升 16
三、gamma-tocopherol對蕈毒鹼受器連結鈣訊之影響 17
四、牛血清蛋白對gamma-tocopherol引發[Ca2+]i↑之影響 17
五、gamma-tocopherol對細胞生長的影響 18
六、BSA/gamma-tocopherol之濃度和時間效應 19
七、BSA和gamma-tocopherol形成複合物造成細胞毒性 20
八、BSA/gamma-tocopherol複合物引起細胞凋亡 21
討 論 23
一、gamma-tocopherol具有引發[Ca2+]i↑之能力 23
二、gamma-tocopherol對蕈毒鹼受器之影響 25
三、BSA和gamma-tocopherol形成complex 26
四、BSA/gamma-tocopherol之毒性機制 27
參考文獻 31
圖 表 38
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