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研究生:葉士豪
研究生(外文):Shih-Hao Yeh
論文名稱:細胞內鋅離子對NMDA受器調控之研究
論文名稱(外文):The intracellular regulation by zinc to NMDA receptor
指導教授:張兗君
指導教授(外文):Yen-Chung Chang
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:61
中文關鍵詞:NMDA受器細胞內鋅離子微量注射爪蟾蛙卵
外文關鍵詞:NMDA receptorintracellular zincmicroinjectionXenopusoocyte
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神經突觸是神經細胞間訊息傳遞的主要通道,後突觸膜上的NMDA受器可以被前突觸端釋放之麩胺酸活化,與學習和記憶形成的機制非常相關。鋅離子與麩胺酸共存於前突觸端,並一起釋放至突觸間隙,可由細胞外抑制NMDA受器,也可以透過多種管道進入後突觸細胞。鋅離子在細胞外對NMDA受器的抑制,已經有相當多的研究,但細胞內鋅離子的調節作用,則鮮少有人研究。進入細胞內的鋅離子,是否也可以對NMDA受器進行調節,是本論文研究的主要目標。
我們將大鼠的NR1a/NR2B共表現於爪蟾卵母細胞上,灌流NMDA溶液,利用壓力打藥器作離子注射,將鋅離子打入蛙卵中,觀察細胞內鋅離子對NMDA受器活性的調節作用。我們在離子注射的前後,各記錄一次NMDA誘發電流,在不同濃度鋅離子的注射下,比較前後電流的差異,我們發現注射鋅離子前後,NMDA受器的活性沒有明顯的改變。我們也即時記錄離子注射時蛙卵細胞膜漏電流的變化,分析離子注射對膜電流產生的即時效應。我們對表現NMDA受器的蛙卵注射1 mM~1 M的鋅離子,與在未表現蛙卵注射鋅離子的對照實驗結果比較,我們發現我們所見到的離子效應為鋅離子對蛙卵本身的影響,我們依然沒有見到鋅離子由細胞內對NMDA受器的調節作用。因此我們推論,如果細胞內鋅離子對NMDA受器本身有調節的作用,則此作用非常微小。相對於細胞外鋅離子高效率的抑制,在蛙卵表現系統上,我們未發現細胞內鋅離子對NR1/NR2B受器的直接調節作用。

謝誌 ………………………………………………………………………… i
摘要 ……………………………………………………………………… iii
壹、導論 …………………………………………………………………… 1
1-1. NMDA受器……………………………………………………………… 1
1-2. 鋅離子與NMDA受器…………………………………………………… 6
1-3. 蛙卵表現系統與微量注射…………………………………………… 9
貳、材料與方法 …………………………………………………………… 10
2-1. 質體DNA的轉殖與製備……………………………………………… 10
2-2. cRNA的製備(In Vitro Transcription)………………………… 11
2-3. 蛙卵的取得…………………………………………………………… 11
2-4. 微量注射(Microinjection)……………………………………… 12
2-5. 受器之電生理記錄(Two-electrodes Whole Cell Voltage-clamp)……………………………………………………………………………… 13
2-6. 壓力打藥器(PicoPump)…………………………………………… 14
2-7. NMDA溶液的灌流……………………………………………………… 15
2-8. 電生理訊號的擷取、儲存與分析…………………………………… 16
參、結果 …………………………………………………………………… 17
3-1. NMDA受器次單元的轉殖……………………………………………… 17
3-2. NMDA受器的電生理活性測試………………………………………… 17
3-3. 細胞外鎂離子及鋅離子對NMDA受器的抑制………………………… 18
3-4. 離子微量注射:實驗條件的控制與改進…………………………… 19
3-5. 細胞內注射離子後對NMDA受器活性影響之分析…………………… 24
3-6. 細胞內注射離子對NMDA受器的立即效應之分析…………………… 26
肆、討論 …………………………………………………………………… 28
4-1. 細胞內鋅離子對NMDA受器沒有明顯的作用………………………… 28
4-2. 離子注射實驗的變因………………………………………………… 29
4-3. 其他實驗方式………………………………………………………… 31
伍、參考文獻 ……………………………………………………………… 32
陸、圖表 …………………………………………………………………… 45

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