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研究生:林金郎
研究生(外文):Jin-lang Lin
論文名稱:台灣眼鏡蛇心臟毒素的結構對毒素進入心肌細胞內的影響研究
論文名稱(外文):Effect of cobra cardiotoxin structure on its internalization process in H9C2 cell
指導教授:吳文桂
指導教授(外文):Wen-guey Wu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生物資訊與結構生物研究所
學門:生命科學學門
學類:生物訊息學類
論文種類:學術論文
論文出版年:2005
畢業學年度:94
語文別:中文
論文頁數:54
中文關鍵詞:蛇毒心臟毒蛋白心肌細胞
外文關鍵詞:cobra cardiotoxinCardiomyocyte
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過去的研究已經發現心臟毒蛋白A3會進入H9C2心肌細胞,最終其路徑會進入到粒線體中,並且發現過程會受到膽固醇及溫度的影響(王佳蕙,2005,博士論文)。因此本論文利用Rhodamine螢光標定不同心臟毒蛋白,以共軛焦顯微鏡觀察,研究其它心臟毒蛋白是否會有與A3相同的機制表現。結果顯示心臟毒素A2與A3進入到細胞中的量幾乎差不多,並且遠大於心臟毒素A1和A6,顯示的第一指環區(loop 1)可能會影響心臟毒蛋白進入到細胞的量。而在溫度影響方面,所有心臟毒蛋白皆在低溫下受到抑制完全不進入到細胞中,顯示進入的路徑是跟溫度有很大的關係。膽固醇含量不同的結果則顯示心臟毒素A2和A3有著很明顯完全相反的結果差異,表示出第二指環區(loop 2)對於細胞上膽固醇含量的影響似乎有很大的關係。以上研究結果發現不同的心臟毒蛋白進入到細胞的量是不一致的,但是卻都會受到溫度與膽固醇的影響,由結構序列則可以發現似乎是與心臟毒素上的第一、第二指環區有很大的關係。然而也發現到不同心臟毒蛋白進入到細胞中的路徑也似乎並非一致,不過此部分還需要更進一步的實驗去探討。
It has recently been shown that CTX A3 internalizes into H9C2 cell and targets at mitochondria. It is a temperature-dependent and cholesterol-sensitive process. In order to understand the structure-function correlation of CTX internalization, I employed confocal microscopy to study the internalization of different rhodamine-labeled CTX homologues. My results showed that the amount of internalized CTXs have the tendency of A2 ~ A3 > A1 ~A6. It suggests the ability of CTX internalization may correlate with the loop 1 structure.
In addition, CTX treatments under low temperature impaired their internalization, which implies the possible temperature-sensitive pathway involved.
Finally, the effect of cholesterol content on their internalization is different for CTX A2 and A3, which suggests the loop 2 structure might play important role.
In summary, different cardiotoxins exhibit different behavior as distinguished by either internalized amount or cholesterol sensitivity. Comparison of their primary sequences suggests the loop 1 and loop 2 regions play role in CTX internalization, and the different effects are contributed by their structural diversity.
第一章 緒論-----1
1-1蛇毒(Snake venom)----1
1-2心臟毒素( Cardiotoxin,CTX )簡介----3
1-2-1 心臟毒素的結構----3
1-2-2心臟毒素的生理及藥理作用----4
1-3 心臟毒素A3進入H9C2心肌細胞的調控機制----7
1-4 心臟毒素結構與醣胺素(GAGs)和脂質(lipid)的作用關係----11
1-4-1心臟毒素結構與醣胺素(GAGs)的作用關係----11
1-4-2心臟毒素與脂質(lipid)的作用關係----12
1-5研究目的----15
第二章 實驗材料與方法----16
2-1實驗材料----16
2-2實驗方法----17
心臟蛇毒的製備----17
細胞培養----18
螢光標定----19
Edman degradation----20
固定細胞(Fixed cell)螢光染色----24
4℃下活細胞染色----25
膽固醇含量對心臟毒素進入細胞的影響----25
共軛焦顯微鏡觀察----26
第三章 實驗結果----30
3-1不同心臟毒蛋白進入固定細胞與活細胞的比較----30
3-2 溫度及膽固醇含量對心臟毒素進入細胞的影響----32
第四章 討論----46
4-1不同心臟毒蛋白進入固定細胞與活細胞的比較之探討----46
4-2 溫度與膽固醇含量對不同心臟毒蛋白進入細胞影響之探討----48
4-3 結論----49
參考文獻----50
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