生性 BMPRII homolog 突變果蠅之運動神經元中則可使果蠅生存，並且部分恢復 NMJ 中突觸 (synapse) 的型態及功能(Marques et al., 2002)，另外，在哺乳類 動物的研究中發現當神經受到損傷時，運動神經元中的 BMP2 mRNA 表現量會 增加(Wang et al., 2007b)。
過去曾有多篇研究利用 nerve ligation 實驗方法，觀察內生性蛋白質在軸突 內的運輸情形，用以證明其蛋白質對神經元而言是否具有生理功能。在過去研究 中來自許旺細胞及肌肉的 Glial cell line-derived neurotrophic factor (GDNF)已被 證實為運動神經元的 neurotrophic factor 之一，而在 nerve ligation 實驗發現其 會堆積在打結點近端處位置，顯示其在神經軸突內有順向運輸的現象(Russell et al., 2000)，亦有研究利用 nerve ligation 實驗方法觀察到 BMP6 會堆積在打結點 遠端處的位置，顯示其在軸突內有逆向運輸的情形，且由許旺細胞產生的 BMP6 亦被證實能夠促進運動神經元的存活(Wang et al., 2007a)。另外，在 TGF-beta 2 的研究上發現，許旺細胞以及肌肉皆會表現 TGF-beta 2，而在 nerve ligation 實 驗中觀察到 TGF-beta 2 在打結處的兩端皆有堆積的現象，顯示其在運動神經元
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之軸突內具有雙向運輸的特性(Jiang et al., 2000b)，亦有研究指出在神經撕裂傷 的老鼠身上每天給予 TGF-beta 2 處理，4 週後觀察發現 TGF-beta 2 能夠降低運 動神經元死亡的數目(Jiang et al., 2000a)。而本論文中觀察到 BMP4 會堆積在打 結處的兩端，其運輸方向有順向運輸及逆向運輸(圖八)，其運輸方向與先前研究 發現的 TGF-beta 2 一致(Jiang et al., 2000b)，由這些結果顯示，BMP4 或許與 TGF-beta 2 具有相似生理功能。綜合上述，我們認為周邊肌肉組織及許旺細胞 可能藉由多種因子影響運動神經元各種不同的生理功能。
Muscle-derived factor 對於運動神經元的發育及至成熟穩定非常的重要，曾 有研究發現肌肉發育缺失的老鼠，其 90%的運動神經元會死亡(Oppenheim et al., 1993)，亦有文獻指出在大鼠的肌肉中移植進過度表現 neurotrophin-4 (NT-4) 的 細胞後，會大量增加運動神經末梢的分支 (branching)數目，顯示由肌肉所分泌
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生 (axon regeneration) (Parikh et al., 2011)，且在中樞神經系統損傷後發生去 髓鞘化 (demyelination) 過程中，短時間內給予外源性 (exogenous) BMP4 處 理可以在促進寡突膠前驅細胞 (oligodendrocyte progenitor cells , OPCs)、星狀 膠神經細胞 (astrocyte)以及為神經膠細胞 (microglial) 增生(Sabo et al., 2011)， 態下，BMPRII protein 會表現在神經軸突內(圖一)，BMP4 的 protein 則集中表 現在坐骨神經之許旺細胞上(圖七)，且坐骨神經上能夠偵測到 BMP4 mRNA 表 1990)，然而在神經損傷後的坐骨神經上觀察到內生性 CNTF mRNA 表現量下降，
而由本論文的 nerve ligation 實驗，我們觀察到坐骨神經之 BMP4 mRNA 表現量 有下降的情形(圖九)，另外，由免疫染色實驗我們觀察到 nerve ligation 後的坐 骨神經上 BMP4 則集中在神經軸突內(圖八)。根據上述內容，我們可以知道許旺 細胞在正常的狀態下會產生 BMP4，故 BMP4 是一種 Schwann cell-derived factor，用以維持神經肌肉系統之環境的穩定，而當神經軸突受到損傷時，許旺 細胞所產生的 BMP4 將會被運送進軸突內，參與許旺細胞及運動神經元之間的 機制作用。
過去有研究指出，BMP4 與 CNTF 皆可保護視網膜神經細胞抵抗 NMDA 所
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由本論文之實驗發現，BMP4 可以保護 NG108-15 神經細胞對抗由 glutamate 引 起的毒殺反應。過去曾有研究發現，在卵巢組織培養系統中，加入 BMP4 中和 性抗體抑制掉卵巢內部 BMP4 訊息傳遞路徑，將會促進細胞凋亡反應，然而以 BMP4 處理卵巢組織後則可觀察到其 transforming growth factor-alpha (TGF) mRNA 表現量下降，顯示 BMP4 為卵巢細胞的 survival factor，其可以透過調控 growth factor 而維持組織細胞的穩定(Nilsson and Skinner, 2003)，故 BMP4 也 許可以藉由調控 NG108-15 神經細胞內部 growth factor 的表現而達到神經保護 之效果。另外，曾有文獻指出 glutamate 誘導的興奮性毒殺作用會透過活化 MEK / ERK 訊息路徑促使細胞膜上 AMPA receptor 大量增加，細胞內環境更為興奮 性後引起細胞死亡 (Zhu et al., 2002)，而過去的研究發現 BMP4 在胚胎幹細胞 (embryonic stem cells, ES cells) 內會藉由抑制 ERK/p38 MAPK 訊息路徑達到 細胞自我更新 (self-renewal)，且 p38 MAPK 的抑制劑之功能與處理 BMP4 的 效果相同(Qi et al., 2004)，另外，在大腦缺血性損傷 (cerebral ischemia) 的動 物模型中則發現 p38 MAPK 的抑制劑會降低 glutamate 誘導之細胞死亡反應 (Barone et al., 2001)，所以，BMP4 也許會透過抑制 glutamate 所活化的訊息路 徑以達到保護神經細胞的功能。因此，我們未來可進一步確認 BMP4 保護
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立 政 治 大 學
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Na tiona
l Ch engchi University
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第五章 結論
在本篇論文中以各種不同技術初步證實 BMP4 於神經肌肉系統中具有生理 功能，在正常狀態下，許旺細胞與肌肉皆會產生 BMP4，而由肌肉產生的 BMP4 亦會表現在 NMJ 處，且其 mRNA 及 protein 之表現量皆會受到神經衍生性蛋 白 Agrin 的調控。另外，由 nerve ligation 實驗中發現 BMP4 在神經軸突內有雙 向運輸的現象，當神經受損時，周邊的肌肉組織及神經軸突上的許旺細胞之 BMP4 mRNA 表現量皆受到影響。
此外，我們亦觀察到 BMP4 能夠保護 NG108-15 神經細胞對抗 Glutamate 誘導的興奮性毒殺作用，且在興奮性毒殺時期以及恢復時期皆以 BMP4 處理對 神經細胞的保護效果最好。由本論文之實驗結果推論：BMP4 是運動神經元的 peripheral-derived factor 之一，在神經肌肉系統中具有生理功能，而當神經受 損時，其可能會參與調控運動神經元的存活，故 BMP4 未來也許可以應用在臨 床醫療上，發展成治療運動神經元疾病的方法。
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