一氧化氮合成酶抑制劑在新建立的神經-肌肉細胞共養模式中對突觸後乙醯膽鹼受器聚集之影響

NOS Inhibitor Interfering With Postsynaptic AChR Aggregation In A Novel Nerve-Muscle Coculture Model

吳如恩(Ju-En Wu)

指導教授 ： 陳贊如 ； 陳順勝

高雄醫學大學/醫學院/醫學研究所/碩士(2003年)

摘要

許多關於神經肌肉結合點(neuromuscular junction，簡稱NMJ)的研究是利用細胞培養模式進行，最常見的模式是以NG108-15神經細胞與離體肌肉細胞或肌肉細胞株進行共同培養，但NG108-15為神經細胞瘤(neuroblastoma)與膠細胞(glial cell)雜交後所產生的細胞株，同時兼具兩者的特性，若欲研究單獨神經細胞特性對神經肌肉結合點發育的影響，使用NG108-15將無法排除膠細胞特性參與其中的可能影響，因此本論文使用純粹的神經細胞株(Neuro-2A)與肌胚細胞株(G-8)做共同培養(coculture)。此模式的建立，可提供更為有效的實驗模式，以探討神經細胞在沒有膠細胞存在之情況下參與NMJ形成的機轉。
NMJ形成的過程中，肌纖維膜上乙醯膽鹼受器(acetylcholine receptor, AChR)聚集是一典型的早期現象，而一氧化氮(nitric oxide, NO)會參與AChR聚集的過程已被許多研究證實，因此本實驗在建立Neuro-2A與G-8細胞共養(cell coculture)系統後，分別在不同的培養時間加入一氧化氮合成酶(nitric oxide synthase, NOS)的抑制劑L-NAME，評估在細胞共養不同階段，NO產量受抑制後對於AChR聚集的影響，以觀察NO參與突觸形成過程的時間流程，並可證實新的神經-肌肉細胞共養模式成功建立。
實驗結果顯示，以帶有螢光的α-bungarotoxin標定AChR時，可偵測到與Neuro-2A神經細胞共養的G-8肌管上有AChR的聚集形成，並且隨著共同培養時間增加，具有AChR聚集之肌管數量也會隨著增加。雙重螢光染色結果也發現Neuro-2A的神經突起會延伸中止在G-8肌管表面，並且該位置的肌膜上會有明顯的AChR聚集形成。至於NOS抑制劑的影響方面，將L-NAME分別在細胞共養24小時與72小時後加入培養盤內，再觀察其後不同天數時AChR聚集之影響。結果於細胞共養24小時後的突觸形成早期時，抑制NOS對 AChR聚集無顯著影響，但在共養72小時後，亦即AChR聚集量將達高峰前加入L-NAME，則具有AChR聚集之肌管數量會顯著性大幅減少，此現象顯示AChR在初期只形成面積較小的斑塊狀聚集，此時對NO需並不大；而在稍晚時，小斑塊開始集結形成大面積的AChR聚集，此步驟即需要NO之參與。因此本實驗証實當Neuro-2A細胞引發G-8肌管AChR聚集時，NO的參與可促使大面積AChR聚集的形成，此過程應涉及細胞骨骼蛋白複合物D/UGC（dystrophin/utrophin glycoprotein complex）與較小的AChR聚集間的互相聯結，進而造成較大型的聚集出現。

關鍵字

乙醯膽鹼受器聚集 ； 神經肌肉結合點 ； 一氧化氮合成酶共同培養

並列摘要

Most of our understanding of synapse formation and stabilization comes from extensive studies performed at the neuromuscular junction (NMJ). The analysis of vertebrate NMJ development has been facilitated by experimentation with neuron-muscle coculture model. NG108-15, a clonal neuroblastomaÎglioma hybrid cell line, is frequently used to coculture with muscle cells. However, this hybrid cell line possesses features of both neuroblastoma and glioma. It is well known that glial cells contribute to the formation of synapse, thus a novel neuron-muscle coculture model is needed to eliminate the possibility of contribution from glial cells. In this study, Neuro-2A, a clonal mouse neuroblastoma cell line, is selected to coculture with G-8, a clonal mouse skeletal muscle cell line. This coculture model would provide experimentally advantageous for addressing a wide variety of fundamental issues in neuromuscular synaptogenesis.
During development of the NMJ, clustering of acetylcholine receptors (AChRs) is one of the first signs of postsynaptic specialization and is induced by nerve-released agrin. Recent studies have revealed that several molecules are involved in assembling and stabilizing AChR aggregates and postsynaptic apparatus. Among these, nitric oxide synthase (NOS) has attracted many attentions for it’s multipurpose in the synaptogenesis. Previous investigations have demonstrated that nitric oxide (NO) mediates the agrin-induced AChR aggregation and the downstream signal transduction in the skeletal myotube. In other words, NOS is believed to act as an important factor targeting the aggregation of AChRs. In the present study, an attempt was made to examine the time course of NOS participating in AChR aggregation. NOS inhibitor, L-NAME, was applied to the Neuro-2A/G-8 coculture system to assay the influence on AChR aggregation.
The results in this study showed that, during the 6 days coculturing period, the proportion of myotubes possessing AChR aggregates increased as the coculturing day prolonged. The maximum proportion was reached after 4 days coculturing. Double fluorescent stain showed that Neuro-2A extended it processes and ended onto the surface of G-8 myotube where the AChR aggregation was evident. As for the effects of L-NAME treatment, decrements the formation of AChR aggregation and the size of AChR aggregates were observed in a dose dependent manner while applying the treatment at the end of 3 days coculture. However, no significant effect could be examined while treating L-NAME after 1 day coculture. These data indicated that NO was required for the formation of large AChR aggregates in response to neuron.
In conclusion, the Neuro-2A/G-8 coculture model established in this study is available to investigate the mechanism of NMJ formation. In addition, the results in the present study demonstrate that NO is not only required for the formation of AChR aggregation, but are necessary for stabilization of nerve-induced AChR aggregation.

並列關鍵字

acetylcholine receptor aggregation ； neuromuscular junction ； neuronal nitric oxide synthase ； coculture

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