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標題: | 心臟毒蛋白致死心肌細胞之生化研究:引發粒腺體活性的下降 Snake-venom Cardiotoxin-induced Deactivation of Cardiomyocyte Mitochondria |
作者: | Su-Ling Shih 施夙玲 |
出版年 : | 1991 |
學位: | 碩士 |
摘要: | 心臟毒蛋白對心肌細胞作用時,有一段時間的潛伏期。在此潛伏期,細胞膜仍維持完整時,已引起細胞內鈣離子的增加,腺核?三磷酸的減少。以 MTT比色法分析,發現毒蛋白作用於心肌細胞的潛伏期間,粒腺體還原 MTT的能力下降。因為 MTT 被粒腺體還原的反應是發生在呼吸鏈中某一特殊的部位,此現象可能意味著粒腺體功能受損,亦可能是造成潛伏期中腺核?三磷酸下降的原因之一。以 rhodamine123螢光染色觀察,結果顯示經過毒蛋白處理與未經毒蛋白處理,其粒腺體分佈似乎無顯著差異。細胞若先以適量鈣離子拮抗劑,如 3.75uM flunarizine , 3.75uM diltiazem 或 10uM的verapamil 處理,均不會抑制由毒蛋白引起粒腺體活性的下降。以 2uM 的 ruthenium red 抑制細胞內鈣離子進入粒腺體或內質網則會促進毒蛋白對粒腺體活性的影響。當細胞外鈣離子濃度增高至3uM以上時,細胞內腺核?三磷酸含量,粒腺體還原 MTT 的能力並無顯著差異,但卻明顯抑制了毒蛋白致死心肌細胞的作用,同時也抑制毒蛋白在潛伏期中導致細胞內腺核?三磷酸含量的減少和粒腺體活性的下降。本實驗中也探討毒蛋白對胞外酵素 ecto-ATPase 的影響,結果顯示毒蛋白對 ecto-ATPase 的活性沒有顯著的影響。 There appeared a latent period during the cardiotoxin—induced degeneration of neonatal rat cardiomyocytes. The immediate effects of CTX in the latent period during which the membrane structure remained intact were the increase in [Ca+2] i levels and the depletion of endogenous ATP. Using MTT colorimetric assay, we found that CTX could suppress the reduction of MTT in cardiomyocytes. The result suggested that CTX would impair the mitochondrial activity during the latent period since MTT was believed to be converted by mitochondrial dehydrogenase. The result of the binding of rhodamine 123 to the cells suggested no alteration in mitochondria distribution during the CTX action. Preincubation of the cells with 3.75uM flunarizine, 3.75uM diltiazem or 10uM verapamil did not suppress the toxin's ability to impair the mitochondrial activity. On the other hand, preincubation of the cells with 2uM ruthenium red , an inhibitor for Ca+2 uptake of mitochondria and endoplasmic reticulum , enhanced the toxin—induced mitochondrial degeneration . Increasing extracellular Ca+2 to higer than 3mM did not cause a change in intracellular ATP content or mitochondrial activity of the controlled cells, but suppressed considerably the events associated with the CTX action. Attempt was paid to assess the effect of CTX on ecto-ATPase of cardiomyocytes . We found that CTX did not show considerable effect on the ecto-ATPase activity of cardiomyocytes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75831 |
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顯示於系所單位: | 生化科學研究所 |
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