臺灣博碩士論文加值系統

蛇毒是一種混合物，其內容物也許各司其職也許會互相合作。台灣眼鏡蛇所分泌的毒液中主要包含有神經毒（neurotoxin或cobratoxin），心臟毒素（cardiotoxin）以及磷脂質水解酵素phospholipase A2（PLA2）等。其中心臟毒素含量最大卻不是最致命；作用的目標細胞也不只限於心肌細胞而可涵蓋各種細胞，所以十分有趣卻也十分不清楚其對於細胞的毒殺機制。以前的研究幾乎都認為心臟毒素造成細胞的死因是壞死（necrosis），但心臟毒素會使得胞內鈣離子濃度增加確有可能讓細胞走向另一條死亡之路---細胞凋亡（apoptosis）。本實驗是以心臟毒素A3為藥物處理中國倉鼠卵巢細胞（Chinese Hamster Ovary cell，CHO-K1），發現蛇毒心臟毒素A3處理CHO-K1後造成細胞有sub-G1現象，細胞膜表面醣胺素種類對於心臟毒素A3產生的sub-G1並無影響，心臟毒素A3處理CHO-K1後沒有觀察到DNA ladder發生，也沒有細胞核斷裂現象，只有細胞核似乎有縮小的現象且caspase抑制劑及細胞蛋白合成抑制劑似乎對心臟毒素A3產生的sub-G1現象抑制作用不大。另外以西方點墨法觀察細胞蛋白質量的變化時，發現似乎高濃度A3處理下，非活化態的caspase 3及α-tubulin似乎會分解或流失而量減少；人類淋巴母細胞（Human lymphoblast，TK6）細胞之AIF（apoptosis inducing factor）受到A3影響有量上升的趨勢。但心臟毒素A3對CHO-K1造成的細胞死亡為兼具壞死及凋亡的混合型，也還需要更進一步探討。

Snake venom is a mixture of versatile proteins, they could act independently or cooperatively. The major components of snake venom of Naja naja atra are neurotoxins (cobratoxins), cardiotoxins(CTXs) and PLA2. Among these, the most abundant are CTXs, but they are not the most lethal ones, and the targeting cells can be all kinds of cells in addition to cardiomyocytes. Hence, this phenomenon attracts our attention, but we still do not know the exact mechanism of cellular toxicity of CTXs. According the previous research, the reason for cell death action of CTXs is necrosis, but the intracellular calcium concentration increasing by CTXs treatment might lead cells to another destine — apoptosis.
This thesis is based on the experiment about treatment of Chinese Hamster Ovary (CHO-K1) with CTX A3, we found that after the treatment, cells will go to sub-G1 phase, the types of glycosaminoglycans (GAGs) on the cell surface have no effects on this treatment, no DNA ladder phenomenon and chromatin fragmentation had been observed, we only observed the shrieking of nuclei, but caspase inhibitors and protein synthesis inhibitors had little effects on the sub-G1 phase cells. Besides, when we using Western blotting method to observe the variation of protein quantity, we found in the treatment of high concentration of CTX A3, the amount of pro-caspase 3 andα-tubulin will decrease. And the amount of apoptosis inducing factor (AIF) of Human lymphoblast TK6 cells will increase after treatment of CTX A3. After all these observations, if the effects of CTX A3 on CHO-K1 are mixing type of necrosis and apoptosis still to be studied further.

中文摘要………………………………………………………………..1
英文摘要……………………………………………………………….2
緒論
一、 總論……………………………………………………………….3
二、心臟毒素(cardiotoxin)結構…………………………………..4
三、心臟毒素 (cardiotoxin) 的生理功能現象…………………..4
四、Apoptosis (細胞凋亡)……………………………………….…5
四之一、總論……………………………………………………….5
四之二、細胞壞死與細胞凋亡…………………………………….6
四之三、細胞凋亡之型態改變…………………………………….7
五、 細胞凋亡途徑……………………………………………………..9
五之一、簡介…………………………………………………….…9
五之二、Caspase 分類………………………………………….…10
五之三、Caspase 作用…………………………………………….10
五之四、細胞凋亡的途徑………………………………………….10
六、材料與方法……………………………………………………….13
六之一、細胞株………………………………………………….…13
六之二、化學藥品 ………………………………………………..13
六之三、細胞培養…………..………………………………………14
六之四、藥物處理………………………………………………….15
六之五、細胞DNA含量分析……………………………………….16
（Hypodiploid DNA content analysis）
六之六、細胞群落存活率分析…………………………………….17
（Clonogenic survival assay）
六之七、小片段DNA萃取………………………………………….17
六之八、Trypan blue染色………………………………………..18
六之九、Giemsa 染色…………………………………………….…18
六之十、Hoechst 33258染色………….…………………………..18
六之十一、 西方點墨法分析…………………………….………….19
（Western blotting analysis）
七、實驗結果…………………………………………….………….…21
七之一、醣胺素（Glycosaminoglycan，GAG）
對於細胞凋亡的影響…………………………….……….21
七之二、對於蛇毒產生的細胞毒性分析…………………………..21
七之三、細胞存活率分析…………………………………………..22
七之四、DNA斷裂電泳分析………………………….…………….23
七之五、細胞核濃縮及染色質破裂的分析…………………………23
七之六、Caspase的抑制劑處理分析………………………………24
七之七、細胞蛋白合成抑制劑對於細胞毒殺的影響分析……….24
七之八、以西方點墨法觀察蛋白質變化………………………….25
八、討論…………………………………………………………….…26
九、附圖表…………………………………………………………….30
十、參考文獻………………………………………………………..…49

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