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研究生:王俊麟
論文名稱:PAG蛋白質對三氧化二砷誘發急性前骨髓性白血癌細胞毒性之角色研究
論文名稱(外文):Role of PAG protein in arsenite trioxide-induced acute promyelocytic leukmia cell appotosis
指導教授:黃榮南
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
中文關鍵詞:三氧化二砷急性前骨髓性白血癌細胞
外文關鍵詞:PAGNB4As2O3APL
相關次數:
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砷化物是廣泛存在於自然環境中的毒性物質,跟人類皮膚癌、肺癌及肝癌的發生息息相關。最近的研究中指出,三氧化二砷能有效地應用於血癌的治療。報告中發現三氧化二砷是經由誘發過氧化氫相關路徑來造成急性前骨髓性白血癌細胞凋亡。TPX II為一過氧化酶,然而,TPX II在三氧化二砷誘發急性前骨髓性白血癌細胞凋亡之角色仍是未知的。
本研究的目的是要探討TPX II在三氧化二砷誘發毒性之角色扮演。實驗結果顯示,以三氧化二砷處理NB4及K562兩株急性前骨髓性白血癌細胞,都會誘發TPX II的表現,且呈現時間相關性。因NB4細胞中的TPX II有明顯較低的表現量,故在處理三氧化二砷後,有較K562細胞高的過氧化氫累積。為了更進一步確定TPX II在三氧化二砷所誘發毒性之角色,故我們將哺乳類TPX II基因轉殖到NB4細胞 (NB4-PAG)。而NB4-PAG在處理三氧化二砷後,有較少的過氧化氫累積,且死亡情況降低。此外,同時以MS (glutathione peroxidase inhibitor)及三氧化二砷處理NB4-V及NB4-PAG細胞後,NB4-V細胞的過氧化氫累積及細胞凋亡均較無MS處理明顯增加許多,且在相同條件下,NB4-PAG細胞也能有效抑制三氧化二砷所誘導的過氧化氫累積及細胞凋亡的發生。這些結果顯示細胞中的TPX II可藉由降低過氧化氫累積來減少細胞凋亡。

Arsenic is a common environmental toxicant, which associated with human skin, lung and liver cancers. However, arsenic trioxide has also been employed recently in leukemia therapy. It has been found that arsenic trioxide treatment resulted in promyelocytic leukemia cell apoptosis via a hydrogen peroxide-dependent pathway. Although thioredoxin peroxidase II (TPX II) has been demonstrated to have hydrogen peroxidase function, the role of TPX in arsenic-induced apoptosis in leukemia remained unknown.
The specific aims of this study is to explore the role of thioredoxin peroxidase II (TPX-II, a hydrogen peroxidase) in arsenic trioxide-induced toxicity. Arsenic trioxide treatment induces the expression of thioredoxin peroxidase II (TPX-II, a hydrogen peroxidase) in a time dependent manner in two acute myelocytic leukemia cells, NB4 and K562 cells. The NB4 cells were shown substantially lower levels of TPX-II, and hence higher H2O2 accumulation than that of K562 cells by the treatment of arsenic trioxide. To further explore the role of TPX II in arsenic trioxide-induced toxicity, the NB4 cells were transfected with cDNA encoding the mammalian TPX II proteins (NB4-PAG). The NB4-PAG cells were shown lower H2O2 accumulation and more resistant to death in response to arsenic trioxide treatment. Furthermore, co-treatment with mercaptosuccinic acid (MS, a glutathione peroxidase inhibitor) significantly enhanced the arsenic trioxide-induced H2O2 accumulation and apoptosis in NB4-V cells. Whilst the NB4-PAG cells were apparently accumulated less H2O2 and insensitive to apoptosis under the same treatment. These results suggest that TPX-II plays an important role in preventing cells from apoptosis by lowering intracellular H2O2 accumulation.

目錄
目錄 --------------------------------------------------- I
「圖」目錄 ----------------------------------------------IV
縮寫與全名對照表表 ------------------------------------- V
第一章、緒論
壹、砷化物的簡介 ------------------------------------ 1
1.砷的分佈及來源 -------------------------------- 1
2.砷化物對人類健康的影響與代謝 ------------------ 1
3.砷的細胞及遺傳毒性 ---------------------------- 2
貳、急性前骨髓性白血病 (APL) 的簡介 ----------------- 3
參、急性前骨髓性白血病之治療現況 -------------------- 5
肆、 PAG 的簡介 ------------------------------------- 6
伍、研究動機與目的 ---------------------------------- 8
第二章、材料與方法
壹、細胞培養 ---------------------------------------- 9
1.細胞株 ------------------------------------------- 9
2.細胞培養 ----------------------------------------- 9
貳、實驗方法 --------------------------------------- 10
1.細胞中 PAG 蛋白質表現量的測定 ------------------- 10
1.1 細胞的收集 ------------------------------------- 10
1.2蛋白質的萃取與定量 ------------------------------ 10
1.3 SDS-PAGE膠體電泳分析 (Sodium dodecyl sulfate polyacrulamide gel electrophoresis) ------------------- 11
1.4西方點墨法 (Western blotting) ---------------------- 11
2.細胞的轉殖作用 (Transfection) 及篩選 (Selection) ------ 12
3.細胞內過氧化氫累積量測定 (DCF assay) --------------- 12
4.細胞毒性之測定 ----------------------------------- 13
4.1細胞生長計數 ------------------------------------ 13
4.2微核的觀察 (.Micronucleus determination) ------------ 13
4.3慧星分析 (Commet assay) -------------------------- 13
5.細胞計畫性死亡 (Apoptosis) 之測定 ------------------ 14
5.1細胞週期Sub-G1之檢測與DNA含量分析 ----------- 14
5.2 DNA 階梯 (DNA ladder) -------------------------- 14
第三章、結果
壹、三氧化二砷誘導PAG之表現 ---------------------- 15
貳、NB4轉殖pag基因細胞株之建立及表現量之確認 ----- 16
參、三氧化二砷誘導細胞累積過氧化氫之分析 ------------ 16
肆、三氧化二砷誘導細胞毒性分析 ---------------------- 17
1.生長曲線分析 ------------------------------------- 17
2.慧星分析 ----------------------------------------- 18
伍、三氧化二砷誘發細胞產生計畫性死亡(Apoptosis)之分析
1.細胞週期Sub-G1之檢測與DNA含量分析 ------------- 18
2. DNA 階梯 (DNA ladder) --------------------------- 19
第四章、討論 ---------------------------------------- 20
第五章、參考文獻 ------------------------------------ 24
圖表 ------------------------------------------------- 31
附錄 ------------------------------------------------- 43
「圖」目錄
圖一、三氧化二砷誘導NB4及K562細胞PAG之表現 -------- 31
圖二、三氧化二砷誘導NB4及K562細胞累積過氧化氫之分析 -- 32
圖三、Pag轉殖基因於NB4細胞中之表現 ------------------ 33
圖四、三氧化二砷誘導NB4-V及NB4-PAG細胞累積過氧化氫之分析
--------------------------------------------------- 34
圖五、三氧化二砷對NB4-V及NB4-PAG細胞之毒性分析 ----- 36
圖六、GPx對三氧化二砷誘導NB4-V及NB4-PAG細胞累積過氧化氫之影響 ------------------------------------------- 37
圖七、GPx對三氧化二砷誘導NB4-V及NB4-PAG細胞之毒性分析
-------------------------------------------------- 38
圖八、三氧化二砷對NB4-V及NB4-PAG細胞毒性之慧星分析 - 39
圖九、三氧化二砷誘發NB4-V及NB4-PAG細胞計畫性死亡之分析
-------------------------------------------------- 40
圖十、GPx對三氧化二砷誘導NB4-V及NB4-PAG細胞計畫性死亡之影響 --------------------------------------------- 41
圖十一、三氧化二砷誘導NB4-V及NB4-PAG細胞產生DNA ladder
------------------------------------------------- 42

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