臺灣博碩士論文加值系統

細胞凋亡為多細胞生物清除不需要及損傷細胞的一個過程。當生物體受到外來刺激，如UV照射或藥物誘導，造成細胞中的DNA損傷。生物體則啟動細胞凋亡將損傷細胞清除。另外細胞凋亡也參與胚胎發育過程中生物個體的型態雕塑
，並且生物體當中新生細胞及凋亡細胞需處於平衡狀態。許多疾病已被證實和新生細胞及凋亡細胞狀態失衡有關，如神經退化性疾病、免疫缺陷、自體免疫反應和癌症。因此生物體中細胞凋亡的研究則越來越受到重視。常用的體外偵測細胞凋亡的方式如TUNEL或利用核酸染劑acridine orange，而偵測生物體中細胞凋亡的方式則較少被提出。因此我們設計一段全名為apoptosis cell death (ACD)的基因序列，用以偵測體內細胞凋亡現象。此序列藉由beta-actin promoter去啟動基因表現，後面接上Nitroreductase(NTR)基因。利用NTR可將metronidazole(MTZ)轉為細胞毒素的特性，用以促進細胞凋亡。中間藉由2A3分割，後段再接上secretion signal結合Annexin 5 (secA5)。利用其緊密結合於凋亡細胞外翻的磷脂質外膜的特性，作為細胞凋亡的指標。我們將此序列顯微注射進入斑馬魚胚胎表現。之後篩選出帶有此基因的F0種魚，並和PL斑馬魚交配產下F1子代。再由F1自交產下具有homozygous的F2子代，利用F2進行後續實驗。利用可促進細胞凋亡的藥物如camptothecin、rotenone、colchicine、amitriptrline、mtz進行投藥處理，或處以UV照射和heat shock。處理後觀察螢光表現可看到綠色螢光成點狀標記凋亡細胞。因此利用斑馬魚表現ACD序列可作為生物體中細胞凋亡的模式生物，有利於之後細胞凋亡之相關研究。

Multicellular organisms to eliminate unneeded and damaged cells in a process by apoptosis. When the organism to external stimuli, such as UV irradiation or drug-induced, resulting in cell DNA damage. Organism will initiate apoptosis to clear damaged cells. In addition, apoptosis during embryonic development are also involved in the pattern of individual organisms sculpture, and new cells and apoptotic cells must be in equilibrium in the organism. Many diseases have been linked to new cells and apoptotic cells state imbalances, such as neurodegenerative diseases, immunedeficiency, autoimmune reactions and cancer. Therefore, the study of apoptosis is more and more attention. Commonly used in vitro apoptosis detection methods such as TUNEL or utilize nucleic acid dye acridine orange, while the detection of apoptosis in live animals is less to be raised.Therefore, we have designed a full name apoptosis cell death(ACD) gene sequences to detect apoptosis in vivo. The sequences is drived by β-actin promoter, connect with Nitroreductase (NTR) gene behind promoter.Using Nitroreductase (NTR) can convert Metronidazole (Mtz) into cytotoxic to promote apoptosis. The middle splited by 2A3, and connect with secA5 behind 2A3. Using Annexin V binds phospholipids with high affinity, as the indicator of apoptosis. We microinjected this sequence into zebrafish embryos. And screening with this gene of fish (F0),which mating with PL zebrafish to produce progeny (F1). Then by F1 self-cross to produce with homozygous F2, and using F2 for subsequent experiments. Using can promote apoptosis drugs such as camptothecine,rotenone,colchicine,amitriptyline and metronidazole for dosing tretment,or impose a UV irradiation and heat shock.Fluorescence was observed after treatment performance can be seen as punctate green fluorescent labeled apoptotic cells. Thus, the use of zebrafish performance ACD sequence can be used as a model organism,facilitate the subsequent apoptosis of related reserch.

謝辭 i
摘要 ii
Abstract iii
目錄 iv
圖目錄 v
表目錄 vi
壹、序論 1
(一) 動物模式物種—斑馬魚 1
(二) 細胞凋亡 1
(三) 細胞凋亡之路徑 2
(四) 細胞凋亡之調控 2
(五) β-actin-ACD construct簡介 5
(六) 研究動機 7
貳、材料與方法 8
(一) 材料 8
(二) 實驗方法 11
參、實驗結果 19
一、β-actin-ACD (β-actin apoptosis cell death) 19
二、利用核酸引子確認構築體的表現 19
三、β-actin-ACD轉基因斑馬魚建立 19
四、利用細胞凋亡誘導藥物觀察Annexin V (A5)的螢光表現 19
五、利用環境壓力誘導細胞凋亡並觀察Annexin V (A5)的螢光表現 20
六、利用β-actin-ACD轉基因魚誘導細胞凋亡觀察細胞凋亡相關基因表現情形 20
肆、討論 21
伍、參考文獻 22
附圖…………………………………………………………………..........................26
附表…………………………………………………………………………………..39

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