酸敏感受體TDAG8與ASIC3參與在酸誘發的機械性痛覺過敏感現象

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戴士評(Shih-Ping Dai) 查詢紙本館藏

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生命科學系

論文名稱

酸敏感受體TDAG8與ASIC3參與在酸誘發的機械性痛覺過敏感現象
(Involvement of proton-sensing receptors TDAG8 and ASIC3 in acid-induced mechanical hyperalgesia)

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摘要(中)

目前在臨床上認為神經受傷、癌症及關節炎所引發的慢性疼痛是一個很嚴重的問題。而這些發炎的組織所造成的酸化現象被認定為慢性疼痛的重要因子。雖然有肌肉發炎疼痛模式相關研究認為ASIC3及TRPV1參與在hyperalgesic priming上，然而相關機制仍不清楚。在這項研究中，我使用小鼠腳掌皮下注射二次酸模式來探討ASIC3、TRPV1及TDAG8在hyperalgesic priming的功能。實驗結果發現，二次酸注射於野生型小鼠將誘發一個長期 (13天) 的機械性痛覺過敏感現象。而ASIC3-/-及TRPV1-/-小鼠在二次酸注射後縮短痛覺敏感現象的時間。另外，TDAG8則參與在酸所誘發痛覺敏感現象的起始階段中。其中，我們從小分子化合物中發現NSC745885與NSC745887能有效抑制酸、完全弗氏佐劑及神經受損所誘發的痛覺過敏感。更發現NSC745885能有效抑制TDAG8的基因表現及功能。因此，從我實驗結果中可以推論，TDAG8參與在起始痛覺敏感性上，而ASIC3及TRPV1參與在hyperalgesic priming。

摘要(英)

Chronic pain induced by nerve injury, cancer and arthritis is a serious clinical problem disturbing many people. Tissue acidosis is a major factor contributing to chronic pain. Although, a previous study in muscle pain model suggested that ASIC3 and TRPV1 are involved in hyperalgesic priming. However, the detailed mechanism for hyperalgesic priming or other factors involved in priming remains unclear. In this study, I used intraplantarly dual acid injection to explore the roles of ASIC3, TRPV1, and TDAG8 in hyperalgesic priming. Dual acid injection in wild-type mice induced a long-term (13 days) mechanical hyperalgesia. Both of ASIC3-/- and TRPV1-/- mice shortened mechanical hyperalgesia after dual acid injection. TDAG8 was involved in the initiation of mechanical hyperalgesia induced by acid. We also found that small molecule compounds, NSC745885 and 887 inhibited mechanical hyperalgesia induced by acid, CFA, or nerve injury. NSC745885 specifically inhibited TDAG8 expression and function. Accordingly, TDAG8 is involved in the initiation of hyperalgesia, whereas ASIC3 and TRPV1 are involved in hyperalgesic priming.

關鍵字(中)

★ 酸敏感受體

關鍵字(英)

★ Proton-sensing receptors

論文目次

中文摘要 Ⅰ
Abstract Ⅱ
致謝 Ⅲ
本文目錄 Ⅳ
圖目錄 Ⅷ
表目錄 Ⅹ

本文目錄
第一章緒論 1
1.1 慢性疼痛 (Chronic pain) 2
1.1.1 神經性疼痛 (Neuropathic pain) 2
1.1.2 發炎性疼痛 (Inflammatory pain) 3
1.2 預發炎模式 (Hyperalgesic priming) 4
1.2.1 二次酸注射誘發肌肉發炎性疼痛(Dual-acid injection induced muscle
pain) 5
1.2.2 酸敏感受體 (Proton-sensing receptors) 6
1.2.2.1 TDAG8 (T-cell Death-Associated Gene 8) 6
1.2.2.2 酸敏感離子通道3 (Acid-sensing ion channel 3, ASIC3) 7
1.2.2.3 辣椒素受體1 (Transient receptor potential vanilloid channel 1,
TRPV1) 8
1.3 研究動機與目的 8

第二章材料與方法 9
2.1 實驗材料 10
2.1.2 實驗動物 (Experimental animals) 10
2.1.2 細胞株 (Cell lines) 10
2.1.3 核醣核酸干擾 (RNA interference) 10
2.1.4 實驗藥品 (Experimental drugs) 10
2.1.5 實驗器具 (Experimental tools) 11
2.2 實驗方法 (Experimental methods) 11
2.2.1 小鼠基因型的判定(Genotyping) 11
2.2.2 基因表現 (Gene expression) 12
2.2.3 質體DNA置備 (Midi-prep.) 13
2.2.4 瓊酯膠電泳製備及電泳分析(Preparation and electrophoresis of agar gel) 14
2.2.5 注射酸引發傷害性疼痛模式 (Acid model) 14
2.2.6 注射CFA引發發炎性疼痛模式 (CFA model) 14
2.2.7坐骨神經慢性壓迫損傷引發神經性疼痛模式 (Chronic Constriction
Injury model, CCI model) 15
2.2.8 機械性痛覺行為實驗 (Mechanical behavior test) 15
2.2.9 繼代細胞培養 (Cell subculture) 16
2.2.10 玻片的前處理 (Coating cover slips) 16
2.2.11 鈣離子成像分析法 (Cell experiments- calcium imaging assay) 16
2.3 統計分析 (Statistics) 18

第三章結果 19
3.1 酸注射誘發機械性痛覺過敏感 20
3.2 酸注射後1小時TDAG8與ASIC3基因表現量增加 20
3.3 二次酸注射誘發慢性機械性痛覺過敏感 21
3.4 ASIC3參與在二次酸注射誘發的機械性痛覺過敏感 21
3.5 TDAG8參與在酸注射誘發機械性痛覺過敏感的起始階段 22
3.6 降低TDAG8基因表現量於酸注射1小時酸敏感受體的基因表現量變化 23
3.7 蛋白激酶A及蛋白激酶Cε參與在酸注射誘發的機械性痛覺過敏感 23
3.8 NSC745885與NSC745887抑制單次酸注射誘發的機械性痛覺過敏感 24
3.9 NSC745885抑制TDAG8表現細胞在酸刺激下引發鈣離子釋放的情形 25
3.10 NSC745885抑制酸注射引發TDAG8基因表現量增加的情形 25
3.11 NSC745885抑制二次酸注射誘發的機械性痛覺過敏感 26
3.12 NSC745885與NSC745887降低完全弗氏佐劑引發的機械性痛覺過敏感 26
3.13 NSC745885與NSC745887降低坐骨神經壓迫引發的機械性痛覺過敏感 27

第四章討論 29
4.1 二次注射酸誘發機械性痛覺過敏感 30
4.1.1 氫離子為酸注射引發痛覺過敏感的主要原因 30
4.1.2 二次酸注射誘發痛覺持續時間長短之差別 30
4.1.3 酸注射誘發單側與雙側痛覺敏感性之差異 30
4.2 ASIC3參與調控周邊酸注射誘發的痛覺過敏感 31
4.2.1 施打APETx2與ASIC3-/-小鼠在抑制酸注射誘發痛覺敏感性的　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
差異 31
4.3 TDAG8參與調控周邊酸注射誘發痛覺過敏感 32
4.3.1 降低或剔除TDAG8基因表現量只抑制酸誘發後2小時痛覺敏感性的
原因 33
4.4 NSC745885與TDAG8的關聯性 34
4.5 NSC745885與NSC745887在發炎性及神經性疼痛的止痛效果 34
4.6 總結 35

第五章參考文獻 36
附錄Ⅰ 70
附錄Ⅱ 73
附錄Ⅲ 77

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指導教授

孫維欣(Wei-Hsin Sun)

審核日期

2015-6-8

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