許多研究證據顯示第三型酸敏性離子通道(acid-sensing ion channel 3, ASIC3) 和瞬態感受器電位陽離子通道(transient receptor potential vanilloid 1, TRPV1)可能參與慢性疼痛的發生，但是對於ASIC3和TRPV1在慢性疼痛中的作用機制仍不清楚。因此我採用Sluka實驗室研發的非發炎性肌肉疼痛小鼠模式，探討ASIC3和TRPV1在慢性疼痛中扮演的作用角色。在野生型小鼠單側腓腸肌注射pH 4.0生理食鹽水會誘發雙側腳掌短期機械性痛覺過敏化；五天內在同側腓腸肌注射pH 4.0生理食鹽水會誘發持久性痛覺過敏化。由此推論，初次的酸性食鹽水注射可能會誘發肌肉痛覺感受器(nociceptors)產生痛覺過敏化促發作用(hyperalgesic priming)。因此，小鼠接受第二次酸性食鹽水注射後，得以產生持久性的慢性肌肉痛覺過敏化。我設計了ㄧ系列的藥理實驗來驗證ASIC3和TRPV1的活化，是否會影響到肌肉痛覺感受器促發作用的產生。我發現活化ASIC3不僅會增強河豚毒素敏感型電壓門控鈉離子電流(TTXs INaV) ，同時也會影響促發作用的持續時間(duration of priming)。相對而言，活化ASIC3或TRPV1均可增加河豚毒素不敏感型電壓門控鈉離子電流(TTXr INaV) ，並與促發作用的建立(establishment of priming)相關。另外，我也發現電壓門控鈉離子通道Nav1.8跟慢性痛覺過敏化維持相關。因為痛覺感受器遭受刺激所釋放的神經胜肽物質P (substance P, SP)會調控中樞神經系統痛覺傳遞並造成周邊神經系統產生神經性炎症反應(neurogenic inflammation)，所以我想進一步探討SP在酸誘發之慢性肌肉疼痛扮演何種角色。令人訝異的是，我發現SP在肌肉的作用截然不同於以往的認知。肌肉酸性注射造成痛覺感受器釋放SP， SP具有鎮痛作用(antinociceptive effect)可以抑制酸誘發之痛覺過敏化。

Increasing evidences suggest that both acid-sensing ion channel 3 (ASIC3) and transient receptor potential vanilloid 1 (TRPV1) are involved in the process of chronic muscle pain, but their precise role in chronic muscle pain remained elusive. A rodent model of non-inflammatory muscle pain was established by Sluka and colleagues. In the pain model, an acid-injection into one side of the gastrocnemius muscle caused a transient mechanical hyperalgesia on both hind paws and the hyepralgesia declined in 24 hours; the bilateral secondary mechanical hyperalgesia became long lasting after a second acid injection within five days in wild type mice. Here, I found the first acid-injection also induced the hyperalgesic priming of muscle nociceptors to trigger the development of long-lasting chronic hyperalgesia induced by second acid-injection. The activation of ASIC3 or TRPV1 is critical for the hyperalgesic priming of muscle nociceptors. Activation of ASIC3 was required for enhanced activity of tetrodotoxin (TTX)-sensitive voltage-gated sodium channels and the duration of priming that determines how long the primed state can stay. However, enhanced activity of TTX-resistant voltage-gated sodium channels with ASIC3 or TRPV1 activation was required for the establishment of priming that determines how long the long-lasting hyperalgesia can be maintained. Moreover, I found Nav1.8 is involved in the maintenance of chronic hyperalgesia. Because neuropeptide substance P (SP) released from nociceptors mediates pain transmission centrally and neurogenic inflammation peripherally, I probed the role of SP in acid-induced chronic muscle pain. Surprisingly, I found acid-induced SP release mediated an antinociceptive effect in muscle nociceptors.

目錄
正文目錄.....................................................................................................I
表目錄......................................................................................................IV
圖目錄.......................................................................................................V

正文目錄
中文摘要..................................................................................................VI
英文摘要...............................................................................................VIII
1. 緒論.......................................................................................................1
1.1 前言..................................................................................................2
1.2 慢性肌肉疼痛..................................................................................2
1.3 第三型酸敏性離子通道和肌肉疼痛相關......................................4
1.4 瞬態感受器電位陽離子通道和肌肉疼痛相關......................6
1.5 物質P和疼痛之關連......................................................................7
1.6 促發作用與急性疼痛轉變為慢性疼痛之作用機轉相關..............8
1.7 研究目的........................................................................................10
2. 實驗材料與方法.................................................................................11
2.1 實驗動物........................................................................................12
2.2 慢性肌肉疼痛之動物模式............................................................12
2.3 機械性痛覺過敏化及熱痛覺過敏化之行為測試........................13
2.4 血漿外滲實驗................................................................................14
2.5 背根神經節神經元細胞之初代培養............................................15
2.6 全細胞膜片箝制記錄....................................................................15
2.7 酸誘發之鈉離子電流....................................................................16
2.8 電壓門控鈉離子電流....................................................................17
2.9 免疫螢光染色................................................................................18
2.10 統計分析........................................................................................18
3. 實驗結果.............................................................................................20
3.1 酸敏性肌肉傳入神經元之分群....................................................21
3.2 周邊TRPV1參與肌肉重複酸性注射誘發之慢性痛覺過敏化 21
3.3 ASIC3參與痛覺過敏化之促發作用............................................22
3.4 痛覺過敏化促發作用之持續與ASIC3和TRPV1相關..............23
3.5 在肌肉痛覺感受器中，活化ASIC3或TRPV1會增強河豚毒素敏感型(TTXs)和河豚毒素不敏感型(TTXr)電壓門控鈉離子電流 (INaV) ..............................................................................................25
3.6 表現ASIC3的肌肉傳入神經元之分群........................................25
3.7 Nav1.8和PKCε在酸誘發的慢性痛覺過敏化中扮演之角色.....27
3.8 失去物質P的訊息傳遞之作用機轉會加速酸誘發慢性痛覺過敏化....................................................................................................29
3.9 物質P可以阻止酸誘發之慢性痛覺過敏化產生.........................30
3.10 肌肉酸性注射釋放之物質P不會造成神經性炎症反應.............31
3.11 物質P會誘發非ASIC3、TRPV1相關的延長性鎮痛作用.......31
4. 討論.....................................................................................................33
4.1 酸誘發肌肉痛覺感受器促發作用可以經由不同模式所活化....34
4.2 促發作用的持續和促發作用的建立是經由不同機制................35
4.3 ASIC3、TRPV1、Nav1.8在慢性肌肉疼痛中扮演不同作用角色
........................................................................................................36
4.4 PKCε不參與肌肉酸性注射誘發之促發作用和慢性疼痛過敏化
........................................................................................................37
4.5 表現IB4或TRPV1肌肉傳入神經元之細胞分群......................38
4.6 物質P在不同組織作用效果不同.................................................39
4.7 酸誘發鎮痛機制之作用................................................................40
4.8 結論................................................................................................40
參考文獻..................................................................................................68
 
表目錄
表1 抑制ASIC3與TRPV1會影響重複肌肉酸性注射造成之痛覺過敏 化的維持.........................................................................................42
表2 利用電生理實驗定性分群不同的肌肉傳入背根神經節神經元上ASIC3與TRPV1的表現.................................................................43
 
圖目錄
圖1 在小型或中型之肌肉傳入背根神經節神經元中，電生理紀錄所得之五種不同類型酸誘發電流曲線..................................................44
圖2 周邊TRPV1參與肌肉酸性注射誘發之機械性痛覺過敏化........46
圖3 ASIC3與TRPV1參與肌肉痛覺感受器之痛覺過敏化促發作用48
圖4 酸會誘發肌肉產生延長性鎮痛作用..............................................50
圖5 在酸誘發之肌肉疼痛模式中，活化ASIC3與TRPV1對於電壓門控鈉離子電流之影響.....................................................................52
圖6 Nav1.8與IB4在肌肉傳入神經元中表現之比例.........................54
圖7 Nav1.8在酸性誘發之慢性肌肉疼痛中所扮演之角色.................55
圖8 在酸性誘發肌肉疼痛模式中，PKCε不參與痛覺感受器促發作用.....................................................................................................56
圖9 SP訊號傳遞對於肌肉酸性注射誘發機械性痛覺過敏化之影響.....................................................................................................58
圖10 SP與ASIC3在酸誘發慢性痛覺過敏化所扮演之角色................60
圖11 酸誘發之SP釋放不會造成肌肉組織神經性炎症反應..............62
圖12 在酸誘發的慢性肌肉疼痛模式中，SP媒介酸所誘發的延長性鎮痛作用............................................................................................64
圖13 離子通道媒介之肌肉痛覺感受器促發作用圖示說明................66

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