近年來，由海洋生物衍生而得的胜肽應用於新藥開發已被視為具有相當的潛力。最近我們發現海洋生物衍生胜肽PCD-1具有生物活性，在先前的研究顯示，它具有抗發炎的特性。神經發炎對於神經病理性疼痛扮演著相當重要的角色，目前並沒有有效的藥物能夠治療神經病理性疼痛。在本研究中，我們分析PCD-1對於大白鼠慢性窄縮性損傷引起神經病變的鎮痛和抗神經發炎的作用。實驗結果發現，當椎管給予PCD-1於慢性窄縮性損傷引起神經病變的大白鼠對於thermal hyperalgesia、mechanical allodynia、weight bearing及cold allodynia等疼痛行為皆有顯著抑制之效果，並且呈現劑量依賴效應。此外，亦可抑制因慢性窄縮性損傷而導致的脊髓神經發炎。在這些實驗結果的基礎上，PCD-1是有相當大的潛力可以被用來做為一個神經病理性疼痛的治療劑。

In recent years, marine organism-derived peptides have been identified as new and potential sources for drug development. Recently, we found that the marine organism-derived peptide, PCD-1 was biologically active, and previous studies have indicated that it have anti-inflammatory properties. The neuroinflammatory processes are known to play a critical role in the development and maintenance of neuropathic pain, for which no effective drugs are currently available. In the present study, we investigated the anti-analgesic and anti-neuroinflammatory effects of the PCD-1 on chronic constriction injury-induced neuropathy in rats. We found that intrathecal injection of PCD-1 produced a significant and dose-dependent inhibition of thermal hyperalgesia, mechanical allodynia, weight bearing, and cold allodynia in neuropathic rats after CCI surgery. Moreover, it also attenuated CCI-induced spinal neuroinflammation. On the basis of these experimental results, we suggest that PCD-1 could be used as a potential therapeutic agent for neuropathic pain.

論文審定書 i
致謝.. ii
中文摘要. iii
英文摘要.. iv
圖目錄 ix
表目錄 xi
中英對照及縮寫表 xii
壹、前言 . 1
疼痛 1
神經病理性疼痛 . 2
神經病理性疼痛致病機轉 6
Glia cell 在神經病變性疼痛之角色 7
神經發炎(neuroinflammation)與神經病理性疼痛關聯性 . 9
現今治療藥物及副作用 . 9
海洋應用於藥物開發利基 .. 13
海洋胜肽來源 16
海洋胜肽種類 17
海洋胜肽之特性與用途 .. 18
海洋胜肽在哺乳類之應用 .. 21
海洋衍生胜肽PCD 家族 . 24
研究動機與目的 24
貳、實驗材料與方法 . 26
實驗細胞與培養 26
小鼠巨噬細胞培養 .. 26
小鼠神經膠質細胞培養 .. 26
動物準備 26
椎管製備 27
試劑藥品 27
抗體 28
儀器 28
離體模式 30
利用LPS 刺激小鼠巨噬細胞表現iNOS 與COX-II 30
利用LPS 刺激glia cell 表現iNOS 與COX-II . 30
利用interferon-gamma (INF-γ)刺激microglia 表現iNOS 與COX-II 30
西方點墨法分析 (Westeron blot asay) . 30
細胞毒殺檢測 31
活體實驗 32
椎管手術 32
坐骨神經結紮誘發神經病理性疼痛模式 32
疼痛行為評估方法 .. 33
脊髓組織樣品收集 .. 38
組織切片及免疫染色步驟 . 38
動物實驗設計及分組 . 39
統計分析 40
參、結果 .. 41
PCD-1 在三種離體模式之抗發炎活性作用 .. 41
PCD-1 對於LPS 誘發小鼠巨噬細胞抗發炎之作用 . 41
PCD-1 對於LPS 誘發microglia 抗發炎之作用 41
PCD-1 對於INF-誘發microglia 抗發炎之作用 .. 42
PCD-1 對於小鼠巨噬細胞和小鼠神經膠質細胞存活率影響 42
PCD-1 對於神經病理性疼痛之作用 43
椎管給予PCD-1 對於CCI 大白鼠之鎮痛劑量依賴效應 . 43
利用疼痛行為評估椎管給予PCD-1 (20 μg)對於CCI 大白鼠之鎮痛作用 .. 43
PCD-1 對於CCI 大白鼠之weight bearing 改變的影響 .. 43
PCD-1 對於CCI 大白鼠之mechanical allodynia 的影響 .. 43
PCD-1 對於CCI 大白鼠之cold allodynia 疼痛行為的影響 44
PCD-1 對於CCI 大白鼠之thermal hyperalgesia 疼痛行為的影響 . 44
椎管給予PCD-1 對於CCI 大白鼠脊髓組織分子之影響 . 45
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域microglia 活化之影響 .. 45
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域astrocyte 活化之影響 . 45
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域TNF-α上升之影響 . 46
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域p-ERK 上升之影響 .. 46
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域p-p38 上升之影響 . 46
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域IL-1β上升之影響 47
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域p-mTOR 上升之影響 47
椎管給予PCD-1 對於CCI 誘發p-mTOR 在microglia、astrocyte 及neuron之影響 47
PCD-1 對於CCI 大白鼠所誘發脊髓背角區域TGF-β1 下降之影響 . 48
椎管給予PCD-1 對於CCI 誘發TGF-β1在microglia、astrocyte 及neuron 之影響 . 48
椎管給予gabapentin 對於CCI 大白鼠之鎮痛的劑量依賴效應 . 49
PCD-1 與gabapentin 的半數有效鎮痛劑量的差異 . 49
PCD-1 對於CCI 大白鼠安全性測試 49
肆、討論 .. 72
PCD-1 之離體抗發炎活性 . 72
PCD-1 抑制CCI 大白鼠之疼痛作用 73
椎管給予PCD-1 對於CCI 大白鼠之脊髓背角訊息分子之影響 75
PCD-1 對於microglia 的影響 75
PCD-1 對於arstrocyte 的影響 . 76
PCD-1 對於TNF-α 的影響 76
PCD-1 對於MAPK 的影響 77
PCD-1 對於IL-1β、TGF-β1 的影響 . 78
PCD-1 對於細胞因子mTOR 的影響 .. 79
PCD-1 抑制神經病理性疼痛之作用探討 .. 80
海洋胜肽PCD-1 未來展望 . 82
參考文獻 .. 83
附錄一、染色拍照分析 95

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