臺灣博碩士論文加值系統

先前的研究發現，磷脂醯膽鹼會造成神經髓鞘剝離退化，並引發感覺疼痛異常，如：觸覺性疼痛和痛覺過敏等現象。本研究藉由磷脂醯膽鹼藥物處理，誘發正中神經局部去髓鞘化，再利用免疫細胞化學分析、動物疼痛行為測試，進一步探討甘丙肽類型2受器在神經病變疼痛中所擔任的機制。首先證實正常大白鼠的第六頸髓段背根神經節表現甘丙肽類型2受器免疫反應神經元為小型神經元，於正中神經4%磷脂醯膽鹼處理一週，背根神經節中甘丙肽類型2受器免疫反應神經元百分比有明顯地增加。進一步以雙重螢光免疫標誌法分析第六頸髓段背根神經節內甘丙肽類型2受器免疫反應神經元是否有神經絲蛋白200, 轉錄活化因子3, 生長相關因子43, 甘丙肽, 神經胜肽Y, 物質P, 神經性一氧化氮合成酶, 辣椒素受器1。結果顯示同時與神經絲蛋白200, 轉錄活化因子3及神經胜肽Y共存的百分比有顯著地增加；但與物質P共同表現的百分比卻是降低。結果顯示磷脂醯膽鹼處理後，誘導甘丙肽類型2受器免疫反應神經元增升部分為大型受傷的神經元且同時具有神經胜肽Y的表現。另外，動物行為測試分析正中神經施以4%磷脂醯膽鹼處理，在動物同側前肢呈現觸覺痛及溫熱覺過敏現象；當其同側前肢掌面皮下注射甘丙肽類型2受器拮抗劑M871或增效劑AR-M1896處理時，分析其行為反應及楔狀神經核內原致癌基因免疫反應神經元的數量變化。結果發現觸覺痛及楔狀神經核內原致癌基因免疫反應神經元數量以M871處理有顯著地緩解並數量下降，而施以AR-M1896有加劇且數量增加。綜合以上結果，推測磷脂醯膽鹼處理誘導甘丙肽類型2受器免疫反應神經元的增升可能透過神經胜肽Y的釋放，而促使楔狀神經核內原致癌基因免疫反應神經元的數量增加，而與觸覺痛訊息的傳遞有所關聯。

Previous studies have revealed that lysophosphatidylcholine could induce nerve demyelination and initiate neuropathic pain, such as allodynia and hyperalgesia. This study used lysophosphatidylcholine inducing the median nerve local demyelination, and then we investigated the role of galanin receptor 2 (GalR2) in neuropathic pain by immunohistochemistry, drug treatment and animal behavioral test. First, we found galanin receptor 2- like immunoreactive (GalR2-IL) production localized in the small-sized C6 dorsal root ganglion neurons of the normal rats, but the amount of GalR2-IL neurons peaked at one week after lysophosphatidylcholine treatment. Furthermore, we used double immunofluorescence of GalR2-IL neurons weather contained NF200, ATF-3, GAP43, Galanin, NPY, SP, nNOS, VR1. The results showed that the percentage of GalR2-IL neurons also labeled NF200, ATF3, NPY were increasing, but decreased in for SP. These results suggested that lysophosphatidylcholine treatment up-regulated GalR2 expression in A-type and injury DRG neurons further containing NPY. After 4% lysophosphatidylcholine treatment in the median nerve, animals developed tactile allodynia and thermal hyperalgesia in treated side of forelimb. Moreover, we employed lysophosphatidylcholine treatment along with GalR2 agonist (AR-M1896) or antagonist (M871) intraplantar application to examine the effect on median neuropathic pain and c-Fos expression in the stimulated side in cuneate nucleus (CN). The mechanical allodynia level and the number of c-Fos-IL neurons in the M871 group were dramatically attenuated, whereas those in the AR-M1896 group had increased compared to the saline group. These results indicated that activation lysophosphatidylcholine -upregulated GalR2-IL neurons may be possibly via promoting NPY release to evoke c-Fos expression in the CN and to transmit LPC-induced tactile hypersensitivity.

致謝 I
論文口試委員議定書 II
中文摘要 1
英文摘要 2
目錄 3
壹、緒論 5
貳、實驗材料和方法 11
1.建立正中神經傷害模式 11
2.灌流動物 11
3.組織製備 12
3.1正中神經 12
3.2第六頸髓段背根神經節 12
3.2.1石蠟包埋處理 12
3.2.2組織貼片處理 13
3.2.3蠟片脫蠟水合處理 13
3.2.4免疫組織化學染色處理 13
3.2.5免疫螢光雙重標誌處理 15
4.藥物處理及動物行為測試 16
4.1動物機械性痛覺敏感刺激測試 17
4.2動物溫熱覺刺激測試 18
5.藥物處理配合電刺激實驗 18
5.1免疫細胞化學法標誌c-Fos免疫反應神經元於楔狀神經核區 19
叁、實驗結果 21
1.GalR2-IL免疫反應神經元在C6 DRG的分布變化 21
2.LPC誘導正中神經去髓鞘的情形 21
3.GalR2與疼痛相關因子在C6 DRG中共同分布比例 21
3.1 GalR2與NF200在C6 DRG中分布情形 21
3.2. GalR2與ATF-3在C6 DRG中分布情形 22
3.3 GalR2與GAP43在C6 DRG中分布情形 22
3.4 GalR2與Galanin在C6 DRG中分布情形 22
3.5 GalR2與NPY在C6 DRG中分布情形 23
3.6 GalR2與SP在C6 DRG中分布情形 23
3.7 GalR2與nNOS在C6 DRG中分布情形 24
3.8 GalR2與VR1在C6 DRG中分布情形 24
4.GalR2免疫反應神經元與在楔狀神經核分布與表現量 24
5.動物對溫熱覺刺激行為試驗 25
6.動物對機械性刺激行為試驗 25
7.術後第七天，掌面注射藥物再施以電刺激，檢測楔狀神經核內的c-Fos免疫反應神經元數量 26
肆、討論 27
伍、附圖 31
陸、參考資料 45

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