神經切斷後會造成眾多分子的改變，像是基因的正向調節及負向調節。先前我們利用Affymetrix arrays的研究中發現，在坐骨神經切斷之後，有許多新穎的基因在表現上有著顯著地變化。在這些基因之中，neuronatin是其中一個表現出正向調節的基因。
在腦部的發育過程中，neuronatin證實與神經的分化有關。它選擇性地表現在新生時期的腦部，而在成年時期的表現量會顯著地下降。目前我們的研究是探討大鼠在坐骨神經切斷後，neuronatin的表現是否和神經性疼痛的症狀有所關聯。
我們利用230-280公克的Sprague-Dawley大鼠來進行實驗。大鼠分為手術對照組與坐骨神經切斷實驗組，於坐骨神經切斷後的第1、3、5及7天收集檢體(L4，L5背根神經節及L4~L5相對應脊髓)。我們運用Randall-Selitto test來觀察大鼠在觸痛行為的改變；運用西方墨點法及免疫組織化學染色法分析蛋白質的變化；運用RT-PCR來研究mRNA量的變化；並且運用神經細胞標記共同定位法來探討neuronatin與疼痛相關的動物行為的關聯性。
我們研究發現，在坐骨神經切斷後，背根神經節中的neuronatin表現量明顯上升。免疫組織化學染色法的結果也顯示在背根神經節細胞中，標記表現neuronatin的細胞數目上升。
神經細胞標記共同定位法的結果顯示，大鼠的背根神經節細胞中，neuronatin主要分佈於未髓鞘化的C-fiber及少量髓鞘化的A-δ fiber中。Neuronatin表現量的上升及神經細胞標記共同定位的結果顯示，neuronatin和觸覺疼痛過敏有關。這種結果指出，在坐骨神經切斷後的機械性觸覺疼痛上升與neuronatin在背根神經元C-fiber及A-δ fiber的表現有顯著地關聯。

Several molecular changes occur following axotomy, such as gene up-regulation and down-regulation. In our previous study using Affymetrix arrays, it was found that after the axotomy of sciatic nerve, there were many novel genes with significant expression changes. Among such genes was neuronatin, whose expression was significantly up-regulated.
Neuronatin was identified as a gene selectively expressed in the neonatal brain and is involved in neuronal differentiation during brain development, and markedly reduced in adult brains. The present study investigated whether the expression of neuronatin correlates with symptoms of neuropathic pain in adult rats with transected sciatic nerve.
Adult male Sprague-Dawley rats weighting 230 to 280 g were used. The rats were grouped into two: those that were sham operated and those that had sciatic nerve axotomy. The specimens-L4,5 dorsal root ganglians(DRG) and their corresponding spinal cords-were collected at post-axotomy day 1, day 3, and day 5. The neuronatin protein contents were analyzed by western blotting and immunohisto- chemistry. Changes in the mRNA levels were evaluated using RT-PCR. Randall and Selitto test was performed to reveal changes in the animal behaviors. The subcellular co-localization of neuronatin with neuronal cell type specific markers were also investigated in correlation with pain-related animal behavior.
It was found that after sciatic nerve injury, the expression of neuronatin in dorsal root ganglians was increased in protein extracts. Furthermore, the results of immunohistochemistry revealed that the cell numbers of DRGs were relatively increased.
Unmyelinated C-fiber and thinly myelinated A-δ fiber in adult DRGs were also among the principal sub-population of primary afferent neurons with distributed neuronatin. The increased expression of neuronatin and its subcellular localization were related to mechanical hyperalgesia. The results indicated that there was a following significant correlation between mechanical allodynia axotomy of sciatic nerve and the increased expression of neuronatin in C-fiber and A-δ fiber of DRG neurons.

論文審定書 i
致謝詞 ii
一. 中文摘要 1
二. 英文摘要 3
三. 背景介紹 6
四. 實驗目的 11
五. 實驗方法 12
動物準備 12
動物實驗-坐骨神經切斷術 12
Randall and Selitto test 13
蛋白質萃取液的製備 14
蛋白質萃取 14
總蛋白質定量 14
西方墨點法 15
RNA萃取 16
反轉錄-聚合酶連鎖反應分析 17
免疫組織化學染色法分析 18
結果分析 19
六. 結果 20
七. 討論 24
八. 未來展望 27
九. 參考文獻 30
十. 圖表 35
十一. 附錄 43

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