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研究生:陳宗杰
研究生(外文):Tsung-Chieh Chen
論文名稱:熱中風後大白鼠神經細胞的形態學變化與細胞色素c、Bcl-2與Bax免疫細胞化學
論文名稱(外文):Morphological Changes and Cytochrome c-, Bcl-2-, and Bax-like Immunoreactivity in Neurons of Heatstroke-induced Rats
指導教授:游祥明
指導教授(外文):Shang-Ming Yu, Ph.D.
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:解剖暨細胞生物學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:81
中文關鍵詞:熱中風細胞色素C免疫細胞化學反應齒狀回海馬回大腦皮質
外文關鍵詞:heatstrokecytochrome cBaxBcl-2dentate gyrushippocampuscortex
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本實驗利用光學顯微鏡、免疫細胞化學染色法與西方墨漬法分析誘發熱中風後大白鼠神經細胞之細胞型態與細胞色素c、Bcl-2與Bax的影響。已有許多研究證據顯示,當熱中風誘發之後,實驗動物腦中發生有大腦局部glucose利用率下降的情況發生,顯示熱中風造成腦內代謝速度減緩。這樣的結果引導許多學者,認為熱中風之後造成腦內氧化壓力的增加,進而對器官功能產生抑制,誘發腦缺血造成神經細胞的傷害。我們利用一套可連續偵測箱內溫度達四小時以上,可以控制溫度的恆溫加熱箱的誘發熱中風模式,將大白鼠麻醉後暴露於環境溫度42℃的環境下並監控其平均動脈壓與肛溫,直到平均動脈壓開始由最高點下降時界定為熱中風生成點。發現熱中風起始時0分鐘,海馬回CA1區細胞形態與假手術動物相似。相較於實驗對照組誘發熱中風後的動物在免疫細胞染色表現上,細胞色素 c表現不明顯,Bcl-2免疫細胞染色表現下降與Bax免疫細胞染色表現則大量表現;齒狀回與大腦皮質上細胞形態出現有大量濃染的神經細胞與細胞間空泡;免疫細胞反應上細胞色素 c免疫細胞染色表現有些表現,而Bcl-2免疫細胞染色表現下降,Bax免疫細胞染色表現則大量表現。熱中風後5分鐘,在海馬回CA1區細胞形態上出現少許的細胞間空泡, Bcl-2免疫細胞染色表現有微量的上升,Bax免疫的免疫染色反應表現下降些許。齒狀回與大腦皮質上細胞形態上濃染的神經細胞與細胞間空泡顯著減少。細胞色素 c免疫細胞染色表現表現降低,Bcl-2免疫細胞染色表現有微量的上升,Bax免疫染色反應表現下降些許。熱中風後10分鐘,海馬回CA1區細胞上出現少數濃染的細胞質並細胞空隙增多,細胞色素 c免疫表現增加,Bcl-2與Bax免疫細胞染色表現有顯著地上升。齒狀回與大腦皮質在細胞形態上,出現少數的泡沫狀構造與皺縮細胞,細胞色素 c免疫細胞染色表現減少,Bcl-2與Bax免疫細胞染色表現顯著上升。熱中風後30分鐘,海馬回CA1區細胞形態上出現濃染的細胞質與細胞空隙,細胞色素 c免疫染色反應表現在整個核周圍,Bcl-2與Bax免疫染色反應表現類似對照表現。齒狀回與大腦皮質在細胞形態上,泡沫狀樹突構造與濃染細胞顯著減少。西方墨漬法分析海馬回上蛋白質,發現誘發熱中風起始時(0分鐘)細胞色素 c增加,隨時間增加降低表現量。誘發熱中風後Bcl-2免疫表現量降低,同時Bax免疫表現則有大量增加的情形出現。結果顯示(1)熱中風誘發大腦神經細胞的損傷。(2)熱中風後神經細胞的損傷情況,隨存活時間的增加而減少。(3)熱中風導致海馬回、齒狀回與大腦皮質的神經細胞走向細胞凋亡。(4)熱中風後細胞質的細胞色素 c大量的增加。(5)Bcl-2與Bax可能參與影響熱中風後神經細胞的存活調控。

Light microscopy, immunocytochemistry and Western blotting were employed in this study for investigating the morphological changes, the efffect of cytochrome c, Bcl-2 and Bax on the rat neurons induced by the heatstroke. Previous evidence indicate that after the heatstroke the metabolism of the glucose decreasesin the brain of the animals. The heatstroke results from the the increase of the oxidative stress and is followed by the ischemic neuronal injury. Sprague-Dawley rats were used in this study. For the pulsatile arterial pressure and heart rate were monitored continuously with a pressure tranducer and a chart recorder (Gould model 2400). Heatstroke was induced by exposing the animal to a high ambient temperature of 430C, the moment at which the mean blood pressure decreased from their peak values was taken as the time of heatstroke onset. At the onset of the heatstroke, the morphological changes were similar to those of the sham-operated rats. At this time, cytochrome c- IR was faintly immunostained, Bcl-2 IR decreased and Bax-IR remarkedly increased. There were vacuoles located among methylene blue darkly staind neurons in the dentate gyrus and cerebral cortex. Cytochrome-c IR slightly increased, Bcl-2 IR decreased and Bax IR remarkedly increased. Five minutes after the heatstroke, there were vacuoles among the neurons, faintly immunostained cytochrome-c IR, slight increase in the Bcl-2 IR and decrease in the Bax IR in the hippocampal CA1 sector. Vacuoles significantly decreased among methylene blue darkly staind neurons in the dentate gyrus and cerebral cortex. Cytochrome-c IR slightly decreased, Bcl-2 IR slightly increased and Bax IR slightly decreased. Ten minutes after the heatstroke, there were a lot of vacuoles among the neurons. Immunostained cytochrome-c IR increased, the Bcl-2 Irand Bax IR markedly increased in the hippocampal CA1 sector. There were a few foamy structure and shrunk neurons, decrease in cytochrome-c IR and increase in Bcl-2 IR and Bax IR. Thirty minutes after the heatstroke, immunostained cytochrome-c IR was found around the nucleus, the Bcl-2 IR and Bax IR remained the same as those in the sham-operated animals in the hippocampal CA1 sector. There were foamy structure and shrunk neurons decreased in the dentate gyrus and the cerebral cortex. Anaysis of Western bloting revealed the icytochrome c IR increased at the onset of the heatstroke and decreased thereafter following the time course. The decrease in the Bcl-2 IR was concomitant with the increase in the Bax IR. Our present evidence indicate that(1)neuron damage was induced after the heatstroke.(2)the neuron damage was gradually decrease from 0 to 30 after the heatstroke induction.(3)the induction of the heatstroke caused the apoptosis in the neurons of the hippocampus, dentate gyrus and cortex. (4)heatstroke induced the increase in the cytochrome c IR accumulated in the mitochondria.(5)Bcl-2 and Bax may be involved in the regulation of the neuronal survival and death after the heatstroke.

中文摘要--------------------------------------------------1
英文摘要--------------------------------------------------3
前言------------------------------------------------------5
材料與方法-----------------------------------------------18
結果-----------------------------------------------------27
討論-----------------------------------------------------37
參考文獻-------------------------------------------------42
圖片與說明-----------------------------------------------53

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