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研究生:蘇弘哲
研究生(外文):Hung Che Su
論文名稱:大鼠視叉上核能量代謝與細胞外的酸化關係調控機制
論文名稱(外文):Energy Metabolism and Extracellular Acidification in the Rat Suprachiasmatic Nucleus
指導教授:黃榮棋黃榮棋引用關係
指導教授(外文):R. C. Huang
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:69
中文關鍵詞:能量代謝酸化視叉上核
外文關鍵詞:metabolismacidificationsuprechiasmtic nucleus
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視叉上核 (SCN) 是哺乳動物約日週期的控制中樞,負責許多生理、生化、行為反應的日夜節律表現。已知SCN 神經元的電活性和代謝活動,都表現日高夜低的約日節律。由於ATP水解與能量代謝產生的氫離子會造成胞外環境的酸化,因此我想使用氫離子選擇性電極,來測量SCN組織的胞外酸化情形(相對於灌流液的pH值)。我的初步結果指出,以35 mM HCO3- 為緩衝的溶液中,SCN胞外酸化程度在白天與夜晚大約都在0.2 pH單位,沒有顯著的差異。但 SCN外圍細胞密度較低的外側與背測區域,則沒有明顯酸化現象。若將緩衝物質改變成10 mM HEPES,則胞外酸化程度增加到 ~0.4 pH單位,同樣還是沒有日夜差異。由於acetazolamide抑制carbonic anhydrase的結果會降低酸化程度約0.15 pH單位,意謂著SCN的胞外酸化是細胞內CO2擴散到細胞外所產生的。以無鉀液或strophanthidin抑制鈉鉀幫浦,會讓胞外酸化程度減少約0.15 pH單位,說明鈉鉀幫浦能量代謝是胞外酸化的重要因素。以NaCN抑制有氧呼吸的狀況下,無鉀液不再能影響胞外的酸化程度,說明鈉鉀幫浦的能量來自有氧磷酸化產生的ATP。有趣的是,以NaCN抑制有氧呼吸就會造成胞外的嚴重酸化。NaCN造成的酸化會被抑制醣質分解作用的iodoacetate (IAA)給完全阻斷,也會被抑制lactate輸送子的藥物給部份阻斷。這些結果說明,有氧呼吸一旦受到抑制,醣質分解的作用便會增強,胞內累積的lactate便會伴隨H+透過輸送子送至胞外,而降低胞外的pH值。IAA抑制能量代謝造成的胞外鹼化作用,約在5分鐘已達穩定,不過IAA的作用是不可逆的。而以無葡萄糖液灌流時,胞外鹼化的作用卻非常緩慢,約需20~30分鐘才到達穩定,而且重新提供葡萄糖時會馬上酸化,說明SCN組織顯然有能量貯存,能在未提供葡萄糖狀況下持續維持能量代謝好一陣子。總而言之,SCN胞外酸化與能量代謝息息相關。至於胞外酸化是否還會影響神經活性,則有待更進一步的探討。
The suprachiasmatic nucleus (SCN) is the central clock in mammals, responsible for daily variations in animal biochemistry, physiology, and behaviors. The SCN exhibits diurnal rhythms in both electrical and metabolic activity, being higher during the day and low at night. Since the production of proton during ATP hydrolysis and energy metabolism leads to extracellular acidifications, I intended to determine extracellular acid shifts in SCN slices using H+-sensitive microelectrodes. My preliminary results indicate an ~0.2 pH units difference (ΔpH) between SCN tissues and the suprefusate buffered with 35 mM HCO3-, but with no day-night variation. No such acidification was observed in extra-SCN areas dorsal and lateral to SCN. The extracellular acid shifts in SCN increased to ~0.4 pH units, again, with no day-night variation, when the buffer of 35 mM HCO3- was replaced with 10 mM HEPES. Inhibition of carbonic anhydrase with acetazolamide produced alkalinization of ~0.15 pH units, suggesting an origin of intracellular CO2 diffusion to extracellular space to cause acidifications. The blockade of Na pump (Na+-K+-ATPase) activity with K+-free solution or the addition of strophanthidin increased extracellular pH by ~0.15 pH units, suggesting that energy metabolism involved with Na pump activity contributes to extracellular acidifications. In contrast, K+-free solution no longer had effect on extracellular pH in the presence of NaCN to block oxidative phosphorylation, suggesting an origin of ATP produced via oxidative phosphorylation as the energy source for Na pump activity. Interestingly, NaCN by itself produced a marked acidification, up to ~0.5 pH units, which was completely prevented with iodoacetate (IAA) to block glycolysis and partially reduced with 4-CIN to block the transport of monocaboxylates. Together the results suggest that accelerated glycolysis in response to the blockade of oxidative phosphorylation produced the large acidification, which was partly due to the outflow of H+ along with lactate via monocarboxylate transporters. Total blockade of energy metabolism with IAA irreversibly increased the extracellular pH within 5 minutes; extracellular alkalinization in glucose-free solution proceeded very slowly, reaching a steady state in 20~30 minutes, but return to 10 mM glucose produced rapid acidifications. The results suggest the presence of energy reserve that allows the SCN to maintain energy metabolism for some time even in the absence of external supply of glucose. In conclusion, my studies with H+-selective microelectrodes indicate a tight relation between energy metabolism and extracellular pH in SCN. It remains to be determined if extracellular acidifications caused by energy metabolism will alter SCN excitability.
目 錄
指導教授推薦書 i
口試委員會審定書 ii
授權書 iii
誌謝 iv
中文摘要 v
英文摘要 vi
目錄 ix簡介 1
1.1視交叉上核 1
1.2自發性動作電位 1
1.3約日節律 2
1.4約日節律和代謝活性 3 1.5 SCN神經與星狀膠細胞間的關係 4 1.6神經細胞外 pH 值 5 1.7能量代謝與氫離子的產生 6 1.8大腦中能量代謝 8 1.9 Astrocyte-neuron lactate-shuttle Hypothesis (ANLSH) 理論 9
第二章 實驗材料與方法 12
2.1.1 氫離子選擇性微電極的置備原理 12 2.1.2 玻璃管準備 13
2.1.3 矽烷化反應 (silanization) 13 2.1.4 氫離子選擇性樹脂 (ion-selective resin) 16 2.1.5 玻璃電極的溶液填充 16 2.1.6 氫離子選擇性微電極校正 (pH calibration) 18
2.2 實驗動物與切片 19
2.2.1 視叉神經上核切片 19
第三章 實驗結果 21
3.1 SCN 組織 pH 值測量 21
3.2 Extra-SCN 的 pH 值測量 22
3.3 SCN 酸化與CO2的產生 22
3.4能量代謝與 SCN pH值變化 23
3.5鈉鉀幫浦與 SCN pH 值變化 26
3.6 SCN 鈉鉀幫浦的ATP來源 27
3.7 SCN 的能量使用 28
3.8 Lactate 對 SCN 組織內 pH 值的影響 29
第四章 討論 31
4.1 SCN 細胞外酸化現象 31 4.2 SCN 細胞外酸化與約日節律 32
4.3 SCN 細胞外酸化與 CO2 33
4.4 SCN 細胞外酸化與能量代謝 33
4.5 SCN 細胞外酸化與鈉鉀幫浦 34
4.6 SCN 細胞外酸化與 lactate 35
4.7 SCN 細胞外酸化與生理意義 35
備註(圖) 38-50
參考資料 51-58
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