臺灣博碩士論文加值系統

神經再生，一直是神經醫學研究中的重要議題。目前對研究周邊神經系統再生已有些相關成果，然而對中樞神經系統損傷後無法再生的狀況，一直是難以解決的問題，導致許多神經相關疾病難以完全治癒，例如：癱瘓、腦中風、神經退化疾病等。而近幾年許多研究顯示這些神經相關疾病中有軸突退化的現象產生，進一步導致神經細胞死亡，因此了解造成受損後軸突退化的機制，成為神經再生中一個很重要的課題。
目前我們實驗室發展出一套神經細胞培養基板裝置（neuronal chip device），使細胞體與軸突生長在不同的特定區域，因此能觀察軸突損傷後細胞體與軸突的變化。本研究分三部份，第一部份觀察不同時間及不同區域的神經細胞經軸突損傷後細胞體與軸突的形態，主要是希望藉由形態上的改變進而了解軸突損傷後對細胞體及軸突的影響。第二部份為觀察不同蛋白質對軸突損傷後細胞體與軸突的影響，利用形態上的改變來探討軸突損傷後軸突退化及軸突再生與不同蛋白質間的交互作用。第三部份為研究軸突蛋白質的表現，我們探討遠端軸突能否有局部蛋白質的新生成，利用click chemistry能以影像方式觀察特定時間點是否有新生成的蛋白質。由初步的實驗結果得知軸突損傷後神經細胞體依舊存活，甚至可以觀察到有新的軸突能延著所轉印poly-L-lysine的細線生長，而受損後遠端軸突也可以偵測到有蛋白質的表現。
目前已知軸突的退化過程包含許多因素，當中可能也會對軸突再生造成影響。由本實驗證明利用神經細胞培養基板裝置能對於研究神經的退化與再生的機制有所助益，最終希望能對神經的退化與再生的機制有更清楚的瞭解，進而對研究中樞神經再生的難題有所助益。

Nerve regeneration is an important issue in neuromedical research. Accumulating evidence has indicated that the axons in adult mammalian brain and spinal cord do not regenerate after injury. On the other hand, in the peripheral nervous system, nerves exhibit remarkable capability of regeneration after injury. It remains rather difficult to cure patients suffering from many diseases wherein the axons in the central nervous systems have been injured. Recent discoveries have also indicated that axonal degeneration occurring in diseased nervous systems would eventually cause neuronal death. Thus, studying the mechanism of axonal degeneration is considered ancillary of studying the mechanisms underlying neuronal regeneration.
More recently, our laboratory develop has developed a neuronal chip device. On the chip surface, axons of cultured rat hippocampal neurons are guided along poly-L-lysine-coated fine lines to grow in areas distinct from where their cell bodies reside. Therefore, we can observe the morphological change and protein expression on cell bodies and axons separately after axotomy.
This study consisted of three parts. The first part consisted of the observations of the morphological changes of cell bodies and axons at different times after axotomy. The second part consisted of studies of the effects of different extrinsic proteins on the degeneration and re-growth of axons after axotomy. The third part focused on analyzing local protein expression in the axon by using Click Chemistry and confocal microscopy. The results indicated that the majority of neurons on the chip could survive axotomy and that, after axotomy and subsequent degeneration of distal axon fragments, new axons could re-grow along the poly-L-lysine-coated fine lines into the areas on the chip designated to be filled by axons.
A number of factors have been known to affect the process of axonal degeneration and regeneration. Here, the study has indicated that chip device developed by our laboratory is a useful tool for studying the mechanisms underlying axonal degeneration and regeneration. The results may help the development of new therapeutic strategies for axonal regeneration in the future.

壹、緒論…………………………………………………………… 1
貳、實驗材料與方法
第一節 實驗材料…………………………………………………6
第二節 實驗方法…………………………………………………8
參、實驗結果
一、不同軸突損傷方式對於神經細胞與軸突的影響…………… 14
二、軸突損傷對較為成熟的神經細胞之影響…………………… 16
三、以免疫染色法觀察軸突損傷後神經細胞及軸突的形態…… 16
四、不同的蛋白質對軸突退化的影響…………………………… 17
五、不同蛋白質對重新生長軸突的影響………………………… 18
六、阻斷蛋白質生成對軸突損傷後神經細胞及軸突的影響…… 20
七、軸突損傷後神經細胞與軸突的蛋白質生成………………… 20
肆、討論
一、不同軸突損傷方式對神經細胞及軸突所造成的影響……… 22
二、探討成長中與較成熟的神經細胞對於軸突損傷後的影響… 23
三、觀察軸突損傷後細胞體與軸突的細胞骨架形態…………… 24
四、探討不同蛋白質對軸突退化與新生長軸突的影響………… 24
五、探討軸突損傷後遠端軸突蛋白質生成的影響……………… 25
六、結語與未來展望……………………………………………… 26
伍、圖……………………………………………………………… 28
陸、參考文獻……………………………………………………… 59
柒、附錄…………………………………………………………… 63

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