肌肉細胞與運動神經在形成突觸的過程中，需要突觸前與突觸後的神經傳遞物質以及滋養因子相互交流以促進突觸的成熟。研究顯示過氧化氫(H2O2)在這個過程中是擔任重要的訊息傳遞分子。H2O2為一種可穿透細胞膜的活性氧化物 (ROS)，它可經由細胞內粒線體呼吸作用、細胞上NADPH oxidase、嘌呤 (purine) 代謝酵素 xanthine oxidase 作用中生成。H2O2較人為常知的是它對於細胞毒性的部分，例如 DNA 損傷、細胞膜的過氧化、細胞老化等，然而其正常生理角色的研究較為少。之前的實驗已證實，H2O2會促進神經的神經自發性傳遞物質乙醯膽鹼 (ACh) 的釋放，而這一個過程與肌肉細胞內所釋放的IGF-1是有關的。先前另一個實驗結果顯示，肌細胞內的H2O2濃度上升和促進神經傳遞釋放有關以及與肌肉細胞內的鈣離子有關。然而很令人好奇的是，H2O2是否造成肌肉細胞內鈣離子的濃度上升，導致IGF-1的釋放，然後進一步影響神經的活性則不是那麼清楚，所以本研究將往這方面探討。
本實驗中，我們利用發育中爪蟾神經-肌突觸細胞培養，以西方墨點法以及單一離子通道電流紀錄方法的方式來探討H2O2促進神經滋養因子IGF (Insulin-like growth factor 1) 釋放分子機制之研究。實驗會從conditioned medium中萃取分泌至細胞外的IGF-1，以西方墨點法來測不同條件下的蛋白質含量，以及利用單通道紀錄去探討不同條件下通道的改變。實驗結果中，H2O2在誘導神經滋養因子釋放時，與細胞內鈣離子濃度的上升是有關係的，當給予不可穿膜的鈣離子螯合劑BAPTA-AM降低細胞內鈣離子濃度之後，發現會降低H2O2的促進作用，此時IGF-1釋放的量減少，顯示肌細胞內的Ca2+的存在與H2O2促進IGF-1的釋放有關，因此，進一步將胞外溶液置換成不含鈣離子的Ca2+-free Ringer之後，阻斷了細胞外鈣離子的來源，實驗結果發現IGF-1釋放的量也減少，這時在不含鈣離子的Ca2+-free Ringer的culture中加入CaCl2後，發現IGF-1釋放的量有顯著回升，由以上結果顯示H2O2促進肌肉細胞釋放IGF-1，是與細胞外的鈣離子流入到細胞內，造成細胞內鈣離子的濃度上升有很大的關係。而進一步的研究顯示，H2O2促進肌肉細胞釋放IGF-1的作用，會因為同時給予H2O2以及ACh而有加強的作用；由於鈣離子進入細胞需經由通道才能進入細胞內，所以當以乙醯膽鹼通道抑制劑氯化筒箭毒鹼Tubocurarine chloride(d-Tc)抑制ACh通道後，ACh的通道會被抑制，使得H2O2無法促進肌肉細胞釋放IGF-1，接著於處理d-Tc之後的culture中加入H2O2，實驗發現也沒有達到促進肌肉細胞釋放IGF-1的效果。結果顯示出H2O2促進肌肉細胞內鈣離子濃度的上升，與ACh通道的開啟有關。接著，單一離子通道的實驗結果表明，ACh通道會自發性的開啟，當給予ACh時ACh通道開啟次數變多，顯示當運動神經靠近肌肉細胞時，所釋放的ACh會促使ACh通道開啟的頻率變高。當ACh促使ACh通道開啟後再給予H2O2之後，實驗發現ACh通道開啟的頻率更高，顯示H2O2會影響ACh通道，讓通道開啟頻繁促使鈣離子由細胞外往細胞內移動，使細胞內的鈣離子濃度增加。由以上結果推斷，H2O2要促進神經滋養因子IGF-1的釋放，會經由H2O2使肌肉細胞外的鈣離子通過ACh通道的開啟而流入肌肉細胞內，使肌肉細胞內的鈣離子濃度上升，進而促進神經滋養因子IGF-1的釋放。
總結實驗結果顯示，神經滋養因子IGF-1在胚胎發育的早期，扮演促進肌肉細胞和運動神經之間突觸形成的重要角色，當運動神經與肌肉細胞形成突觸時，神經突觸會釋放出乙醯膽鹼(ACh)作用於肌肉細胞，使肌肉細胞收縮接著產生過氧化氫(H2O2)進而促進IGF-1釋放。

Successful synaptic transmission at the neuromuscular junction depends on the trophic interaction of the nerve terminals with the postsynaptic specialization of the muscle fiber. We have previously demonstrated that muscle-derived IGF-1 is important in the regulation of spontaneous synaptic transmission at developing neuromuscular synapse. However, the signaling cascades underlying IGF-1 secretion from muscle cells is still a mystery. Our recent results also showed the byproduct of oxidative phosphorylation hydrogen peroxide (H2O2) elicits a muscle-dependent increase in the synaptic activity in 1-day Xenopus motoneuron-muscle coculture and IGF-1 might be the “transducer” of H2O2–induced, muscle-dependent facilitation in the frequency of spontaneous synaptic currents (SSCs). Here we dedicate to explore the role and molecular mechanism of H2O2 in myocyte-derived IGF-1 secretion by measure the IGF-1 protein level in the conditioned medium of day-1 Xenopus myocyte culture using western blotting analysis. Bath application of H2O2 dose-dependently enhances the IGF-1 content in the conditioned medium and reaches its maximal enhancement in IGF-1 secretion at 100 microM. Pretreatment of the culture with Ca2+ chelator BAPTA-AM significantly hampered the H2O2-induced IGF-1 secretion. Moreover, exclusion of Ca2+ from culture medium abolish the IGF-1 content in the medium after H2O2 application, suggesting Ca2+ influx play a crucial role in H2O2-induced IGF-1 secretion. The effect of H2O2-induced IGF-1 secretion was significantly reduced when ACh channel blocker d-Tubocurarine is applied to the medium. The activity of ACh channel recorded by cell-attached patch clamp is dose-dependently increase by ACh and, much to our surprise, the ACh channel open spontaneously even when ACh is absent in the pipette. Furthermore, the frequency of channel opening but not amplitude and dwell time is facilitated by H2O2. Overall, results from this study reveal a reciprocal interaction between presynaptic motoneuron and postsynaptic myocyte which promote the development of neuromuscular synapse. The oxidative phosphorylation byproduct H2O2 increase as the muscle is innervated by motoneuron during early synaptogenesis. The secretion of neurotrophic factor IGF-1 from muscle cell is elicited by H2O2-induced cytosolic Ca2+ rise through facilitation of Ca2+-permeable ACh channel.

論文審定書 i
中文摘要 ii
Abstract iv
圖表目錄 vii
縮寫表 viii
第一章 緒論 1
氧化自由基 (Free radicals) 1
活性氧化物質 (Reactive oxygen species) 2
ROS來源 3
抗氧化系統 5
過氧化氫 (Hydrogen peroxide) 6
過氧化氫對發育成熟的神經-肌突觸之影響 7
IGF-1 (Insulin-like growth factor 1) 8
IGF-1對神經-肌突觸發育初期之影響 9
鈣離子及鈣離子介導信號傳遞 10
鈣離子引發囊泡胞吐作用 10
乙醯膽鹼受體 (Acetylcholine receptor, AChR) 11
研究目的 13
第二章 實驗材料及方法 14
I. 材料 14
II. 試劑儀器及供應商 16
III. 實驗方法 18
A. 電生理紀錄方法 18
B. 西方墨點法 19
C. IGF-1/pDsRed螢光重組質體之製備 24
第三章 實驗結果 30
第四章 討論 39
第五章 參考文獻 45
第六章 附錄 54

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