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博碩士論文 etd-0616106-154849 詳細資訊
Title page for etd-0616106-154849
論文名稱
Title
鈣離子在由cAMP所誘發的神經生長錐偏轉現象的角色之研究
Study on the role of calcium in neuronal growth cone turning induced by cAMP
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
84
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2006-05-30
繳交日期
Date of Submission
2006-06-16
關鍵字
Keywords
神經、鈣離子
cAMP, growth cone, calcium
統計
Statistics
本論文已被瀏覽 5668 次,被下載 2466
The thesis/dissertation has been browsed 5668 times, has been downloaded 2466 times.
中文摘要
當指引因子 (guidance cues) 結合到神經生長錐 (nerve growth cone) 的接受器後,會開啟一連串的訊息傳遞路徑,影響最終目標物─細胞骨架的重新排列,而致使神經生長錐偏轉,指引神經往特定的方向生長。雖然已經發現許多的化學物質都能夠影響神經生長的方向,但確切的訊息傳遞路徑目前為止仍了解有限,尤其是常見的second messenger─鈣離子,在複雜的機制中所扮演的角色,依舊存在互相矛盾的假說及觀點。本實驗主要以爪蟾 (Xenopus Laevis) 的神經細胞培養和turning assay的技術,觀察鈣離子在cAMP所誘發的神經生長錐偏轉現象(cAMP-induced growth cone turning)中的角色為何,以期提供更多證據來釐清鈣離子對神經生長方向的影響。
在前人的研究中,由於turning assay的使用條件不盡相同,因此我們首先必須建立本實驗的turning assay所有條件。在試驗了6種不同的情況後,選取實驗成功率最高,並且呈現穩定偏轉現象的情況,作為之後turning assay的所有條件。確認取細胞培養後14~18小時的culture,以每秒2次,每次20ms duration的給藥方式來進行我們的實驗。
研究結果顯示,當降低了胞內鈣離子濃度後,確實會明顯改變cAMP-induced growth cone turning的現象。並經由前處理不同藥劑得知,從胞內儲存池所釋放的鈣離子,對於偏轉現象亦有影響,若是減低胞外鈣離子內流,則無顯著變化。我們進一步發現,經由ryanodine receptor (RyR) 調控所釋放的胞內儲存池之鈣離子,對偏轉現象的影響較IP3 receptor (IP3R) 所調控的顯著。但在使用Li+來抑制IP3的合成路徑時,意外地看到偏轉角度呈現趨向 (attraction) 和逆向 (repulsion) 兩極化的分布,與使用其他IP3R抑制劑會維持原偏轉現象的結果並不相同。由於Li+會影響其他多條訊息傳遞路徑中的分子,其中包括對神經生長有顯著影響的GSK3-β,因此我們也抑制了GSK3-β,來觀測cAMP-induced growth cone turning是否有所改變,結果發現偏轉現象並未明顯變化。在參考了前人的研究後推論,前處理Li+所造成的特殊神經偏轉現象,可能是多條訊息傳遞路徑交互作用下的結果。
綜合以上所述,我們認為在爪蟾神經細胞培養中,經由RyR調控所釋放的胞內儲存池之鈣離子,對於cAMP-induced growth cone turning的影響顯著,並且認為Li+對於神經生長方向的影響,可能是多條訊息傳遞路徑交互作用下的結果。
Abstract
It is known that guidance cues play important roles in neuronal outgrowth and axonal path-finding. These guidance cues, when bound to the receptors on nerve growth cones, trigger a series of signal transduction pathways that result in dynamic rearrangement of cytoskeleton in neuronal cells. Although enormous studies have been suggested that intracellular calcium concentration is crucial in regulation of neurite outgrowth and cytoskeleton rearrangement, the precise molecular mechanism underlying growth cone guidance is not well understood. The aim of our studies is to set up an evaluation system, growth cone turning assay, which is suitable and reproducible for exploration of the molecular mechanism of guidance cue-induced growth cone turning. The 1-day-old cultured spinal neurons of Xenopus laevis and the approved attractive guidance cue, cAMP, were used in our growth cone turning assay.
We evaluate the effects of neuronal developmental stage, size of pressure, frequency and duration in cAMP-induced growth cone turning. We found application of cAMP with a pressure of 2 Hz in frequency and 20 ms in duration can elicit a reliable growth cone attractive response in cultured spinal motoneurons with 14~18 hours in age. Depletion of intracellular calcium store with thapsigargin in calcium-free Ringer could significantly abolish cAMP-induced growth cone turning. The cAMP-induced growth cone turning was not blunted when calcium was omitted from extracellular fluid or bath application of calcium channel inhibitor suggesting calcium influx is not responsible for the growth cone attractive effect of cAMP. Application of membrane-permeable inhibitors of ryanodine receptors but not inositol 1,4,5-trisphosphate (IP3) effectively occluded the attractive effect elicited by cAMP. The cAMP-induced growth cone turning was unapparent under the presence of Li+, a blocker of IP3 formation. It has been suggest besides block IP3 formation, Li+ is also involved in modulation of several signaling pathways including GSK3-β dependent signaling. However, bath application of GSK3-β inhibitor has no significant effect on cAMP-induced growth cone turning.
Taken together, our results suggest ryanodine-mobilized intracellular Ca2+ store play a major role in cAMP-induced growth cone turning. Moreover, our result on surveying various embryonic stage, drug application protocol also establish a good condition for neuronal growth cone turning assay, which can be used in exploring the molecular mechanism of growth cone path-finding.
目次 Table of Contents
縮寫表 ........................................................................................ 1
中文摘要 ........................................................................................ 2
英文摘要 ........................................................................................ 4
緒論 ........................................................................................ 6
實驗目的 ........................................................................................ 16
實驗材料 ........................................................................................ 17
實驗方法 ........................................................................................ 19
§ Turning assay...................................................................... 19
§ 生長速率測定...................................................................... 20
§ 影像分析及統計.................................................................. 20
§ 藥物前處理.......................................................................... 21
§ 實驗用試劑及供應者.......................................................... 22
結果 ........................................................................................ 23
討論 ........................................................................................ 31
參考文獻 ........................................................................................ 38
圖表 ........................................................................................ 45
參考文獻 References
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