臺灣博碩士論文加值系統

在眼鏡蛇毒液中主要有三種毒蛋白，分別是心臟毒素、神經毒素及磷脂 A2，其中只有心臟毒素的作用目標尚未被證實。根據胺基酸序列中Ser-29及Pro-31的存在與否，及對磷脂質結合能力之不同，可以將心臟毒素分類成S-type及P-type兩種。過去的研究發現，心臟毒素可以引發細胞內鈣離子濃度的增加。再加上許多分子引發細胞凋亡的過程中，也會導致細胞內鈣離子濃度的增加。因此希望藉由以上的訊息，了解心臟毒素是否也會因此而引發H9c2心肌細胞的凋亡，以及S-type和P-type之間對細胞的作用強度是否有差異。本實驗發現，心臟毒素導致細胞死亡的途徑有兩個：一個是細胞壞死，另一個則是細胞凋亡，其中還是以細胞壞死的細胞為主。至於引發細胞凋亡的部分，S-type和P-type之間的作用程度無明顯的分界，唯心臟毒素A4所引發的程度較強。透過流式細胞儀觀察到的Sub-G1 fraction、細胞體積固縮、有次序的DNA斷片電泳圖及Giemsa染色觀察到的核固縮，能夠充分證明細胞凋亡的發生。追蹤心臟毒素A4在早期對細胞的影響，發現它會導致細胞粒線體膜電位下降，以CsA（PTP的抑制劑）前處理，也都未能抑制住膜電位及細胞凋亡的產生。廣效型Caspase抑制劑的前處理，也都未能抑制住細胞凋亡產生。認為心臟毒素A4所引發的細胞凋亡可能不是透過Caspases的途徑。

The venom of cobra could be divided into three type of toxins that is neurotoxin, cardiotoxin and phospholipase A2. Among these three toxins, the target of cardiotoxin is the only one not clarify yet. The cardiotoxin of cobra venom could divided into two type: S-type and P-type, which is differenciate by the existence of P31 and S29, and the binding affinity toward phospholipid. The previous work show that the cardiotoxin will cause the increase in the concentration of Ca2+ in the cytosol. Beside that apoptosis induced by various molecules also to show the same effect of Ca2+ level in cytosol. Therefore, here we perform the apoptosis study induced by the cardiotoxins. We also elucidate the different apoptosis level between S-type and P-type cardiotoxins.
Our result indicated that there are two pathway of cell death induced by cardiotoxins: one is necrosis and the other is apoptosis with the ratio of necrosis is usually higher than apoptosis. The apoptosis phenomena between S-type and P-type showed no significant different, the CTX-A4 perform a slightly higher apoptosis level than other cardiotoxins do. The observation of shrinkage of cell volume, nuclear condensation, sub-G1 fraction, and DNA fragmentation ladder provide us enough evidence of apoptosis occurrence. The early stage of apoptosis effect is the decrease in the mitochondria transmembrane potential. Pretreated with CsA (PTP inhibitor) are proved to be unable to inhibit apoptosis and transmembrane potential decrease. Beside that we also try the broad caspase inhibitor (z-VAD-fmk) which result in no effect of inhibition. This result may be due to the apoptosis induce by CTX-A4 is caspase- independent.

壹、緒論 1
一、心臟毒素 1
（一）蛇毒液 1
（二）心臟毒素的序列及結構 2
（三）心臟毒素的生理影響 4
二、細胞凋亡與細胞壞死的差別 5
（一）形態特徵 7
（二）生化特徵 7
三、實驗目的 13
貳、材料與方法 15
一、材 料 15
二、方法 16
（一）心臟毒素的製備 16
（二）細胞培養 17
（三）細胞存活率檢測 17
（四）流式細胞儀測細胞凋亡 19
（五）電泳觀察DNA斷片 20
（六）Giemsa 染色觀察細胞形態 21
（七）細胞大小 21
（八）粒線體膜電位的變化 22
（九）細胞毒殺率檢測 23
（十）統計分析 25
參、實驗結果 26
一、S-TYPE及P-TYPE對細胞影響的差別 26
二、心臟毒素A4所引發細胞凋亡 26
三、細胞凋亡與細胞存活率的比較 27
四、DNA斷片 27
五、形態改變 28
六、細胞體積的變化 28
七、粒線體膜電位的改變 28
八、短時間細胞凋亡與細胞毒殺的比較 29
肆、討論 30
伍、參考文獻 35
陸、附圖表 51

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