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研究生:朱慶榮
研究生(外文):Ching-Jung Chu
論文名稱:Snail1下游調控基因及其與藥物阻抗性之間的關係
論文名稱(外文):Snail1 downstream target and drug resistance
指導教授:吳國瑞
指導教授(外文):Kou-Juey Wu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
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:63
中文關鍵詞:Snail1藥物阻抗性
外文關鍵詞:Snail1drug resistance
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目前以化學療法治療臨床癌症病患正面臨到嚴重的問題,即是癌細胞對於化療藥物產生阻抗性,但其詳細的分子機轉目前仍未完全明瞭。本實驗室已發表的成果指出,過量表現Snail1的頭頸癌患者接受化學治療的反應較差。因此,本論文主要探討Snail1與化療藥物阻抗性之間的關係。實驗結果顯示當非小細胞肺癌細胞株 (H1299) Snail1的表現量下降時,對於化療藥物cisplatin, paclitaxel會產生敏感性,此時細胞內ERCC1, TUBB3的表現量也下降。在過量表現NBS1的頭頸癌細胞株 (FADU-NBS1) ,發現細胞內的Snail1, ERCC1及TUBB3的表現量也隨之增加。當使用干擾RNA技術,降低此細胞內的ERCC1或TUBB3時,可分別增加對cisplatin, paclitaxel的敏感性。此外以冷光酶試驗系統,發現Snail1蛋白可藉由調控抑癌基因NDRG1的啟動子,影響其表現。由實驗結果推論Snail1的表現量差異相對於化療藥物所產生的反應可能與ERCC1, TUBB3, NDRG1有關。
Chemotherapy resistance is still a major problem for the cancer patients treated with chemotherapeutic drugs. However, the molecule mechanism of chemotherapy resistance is unclear. We previously demonstrated that the head and neck cancer patients with high expression level of Snail1 have poor prognosis. Therefore, we want to discuss the relationship between Snail1 and chemotherapy resistance. The results show that ERCC1, TUBB3 are downregulated and the non-small-cell-lung cancer cell lines (H1299) sensitize to cisplatin and paclitaxel when the expression of Snail1 is decreased. The overexpression of NBS1 in head and neck cancer cell lines (FADU-NBS1) can induce the expression of Snail1, ERCC1 and TUBB3. ERCC1 or TUBB3 is knockdown in NBS1 overexpression head and neck cancer cell lines by siRNA can increase the sensitivity of the cancer cell lines to cisplatin or paclitaxel. Besides, we also find Snail1 can regulate the promoter region of NDRG1, a tumor suppressor gene, and affect the expression of NDRG1. These results indicate Snail1 may mediate the chemotherapy resistance through regulation of ERCC1, TUBB3 and NDRG1 in cancer cell lines.
中文摘要 i
英文摘要 ii
目次 iii
圖次 vi
序論 1
一、 Snail1 1
1. 基因與結構 1
2. 訊息傳遞調控 2
3. 與癌症的關係 2
二、 N-myc downstream-regulated gene 1 (NDRG1) 3
1. 基因與結構 3
2. 訊息傳遞調控 4
3. 與癌症的關係 4
三、 Excision Repair Cross-complementing Rodent Repair Deficiency, Complementation Group 1 (ERCC1) 5
1. 基因與結構 5
2. DNA修復機制 6
3. 與鉑帝爾 (cisplatin) 阻抗性之間的關係 7
四、 Tubulin, Beta 3 (TUBB3) 8
1. 基因與結構 8
2. 微管 (microtubule) 9
3. 與汰癌勝 (paclitaxel) 阻抗性之間的關係 9
研究動機 11
研究目的 13
實驗材料與儀器 14
一、 化學藥品與試劑 14
二、 酵素 15
三、 實驗用抗體 16
四、 套組試劑 16
五、 細胞株 16
六、 菌種 17
七、 質體 18
八、 儀器 18
九、 引子合成訂購:伯森生物科技股份有限公司 18
十、 核苷酸序列定序服務:陽明大學基因體定序中心 18
實驗方法 19
一、 質體 (construct) 建構 19
1. 抽取全基因DNA片段 (genomic DNA) 19
2. 聚合酶鏈鎖反應 (polymerase chain reaction, PCR) 19
3. 限制酵素切割 (restriction enzyme digestion) 19
4. Calf intestinal, CIP 20
5. 去氧核醣核酸片段純化 (DNA fragment purification) 20
6. 質體接合 (ligation) 20
7. 大腸桿菌之轉形作用 (Escherichia coli DH5α transformation) 20
8. 菌體培養 (bacteria culture) 21
9. 小量質體萃取 (plasmid mini-prep) 21
I. pXP2-NDRG1 21
II. pXP2-NDRG1-E box 1 mut 21
III. pXP2-NDRG1-E box 1,2 mut 22
二、 細胞培養 (cell culture) 與轉染 (transfection) 22
1. 細胞培養 (cell culture) 22
2. 短暫性轉染 (transient transfection) 23
3. 穩定細胞株之選殖 (stable clone selection) 23
三、 冷光酶試驗系統 (luciferase assay system) 24
1. 冷光酶試驗 (luciferase assay) 24
2. β-半乳糖苷酶活性檢測 (β-galactosidase activity assay) 24
四、 西方墨點分析法(western blotting analysis) 25
1. 蛋白質萃取 25
2. 蛋白質濃度測定及變性 25
3. 正十二烷硫酸納–聚丙烯醯胺凝膠電泳 (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 26
4. 轉漬 (transfer) 26
5. 偵測目標蛋白 27
五、 細胞存活率分析 27
實驗結果 29
一、 Snail1蛋白藉由NDRG1啟動子調控NDRG1表現 29
二、 Snail1減量表現細胞具有cisplatin, paclitaxel敏感性 30
三、 Snail1減量表現細胞ERCC1, TUBB3表現量明顯下降 30
四、 NBS1過量表現細胞Snail1, ERCC1, TUBB3表現量明顯上升 31
五、 Snail1過量ERCC1或TUBB3減量表現細胞分別具有cisplatin, paclitaxel敏感性 31
討論 33
圖表 36
參考文獻 49
附錄 62
建構質體引子一覽表 62
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