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研究生: 張瑋仁
Wei-Jen Chang
論文名稱: 血清素受體2B參與血清素引起的機械性痛覺過敏
Serotonin receptor 2B is involved in 5-HT-induced mechanical hyperalgesia
指導教授: 孫維欣
Wei-Hsin Sun
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度: 99
語文別: 中文
論文頁數: 86
中文關鍵詞: 血清素血清素受體2B機械性痛覺過敏
外文關鍵詞: hyperalgesia, serotonin, 5-HT, 5-HT2B
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  • 血清素 (Serotonin)是發炎物質的一種,並且主要表現在中樞和週邊神經系統。在受傷或是發炎後,血清素會從血小板或是肥大細胞釋放到週邊神經系統,並引起痛覺或是痛覺過敏。血清素受體共有七個亞型。然而目前還不清楚哪一個血清素受體和血清素引起的痛和痛覺過敏相關。在這篇論文裡,我研究血清素受體2B在血清素引起的痛和痛覺過敏中所扮演的角色。我發現注射血清素或是血清素2的促進劑會產生對機械性刺激的痛覺過敏。而血清素引起的機械性痛覺過敏可以被血清素受體2B/2C的抑制劑給抑制,但血清素受體2A的抑制劑不能抑制血清素引起的痛覺過敏,意指血清素受體2B或2C參與了血清素引起痛覺過敏。考慮到血清素受體2C沒有表現在背根神經節中,血清素受體2B參與血清素引起的機械性痛覺過敏中。進一步我想了解血清素受體2B調控機械性痛覺過敏的機制,我共同轉染了血清素受體2B和與痛覺過敏相關的離子通道(例如酸敏感離子通道家族或辣椒素受體1)到人類胚胎腎臟細胞中,並且以鈣離子攝影驗證血清素受體2B是否會調控這些離子通道。


    Serotonin (5-hydroxtryptamine [5-HT]) is one of the inflammatory mediators present in central and peripheral nervous system. After injury or inflammation, 5-HT is released from platelets and mast cells to peripheral nervous system, causing pain and hyperalgesia. 5-HT receptors include seven subtypes (5HT1-7). However, it remains unclear which subtype of 5-HT receptors is involved in 5-HT-induced pain and hyperalgesia. In this study, we have investigated the roles of 5-HT2B receptors in 5-HT-induced pain and hyperalgesia. We have found that injection of 5-HT or 5-HT2 agonist ?-m-5HT produces significant hyperalgesia to mechanical stimuli. The mechanical hyperalgesia induced by 5-HT injection is inhibited by 5-HT2B/2C antagonist SB206553, but not 5-HT2A antagonist Ketanserin, suggesting that 5-HT2B or 5-HT2C receptors are involved in 5-HT-induced mechanical hyperalgesia. Given that 5-HT2C receptor is not present in not present in DRG, it could be 5-HT2B receptor involved in 5-HT-induced mechanical hyperalgesia. To further understand the mechanism of 5HT2B-mediated mechanical hyperalgesia, we co-transfect 5-HT2B and ion channels that are related to hyperalgesia (such as ASICs or TRPV1) to human embryo kidney 293T cells (HEK293T), and examine whether 5-HT2B receptor regulates these ion channels by calcium image.

    摘要 i Abstract ii 致謝 iii 目錄 iv 圖目錄 vii 縮寫與全名對照表 viii 第一章 緒論 1 1.1 痛覺 2 1.2痛覺的訊息傳導途徑 2 1.3 發炎物質引起的痛覺過敏 3 1.3.1 血清素引起的痛覺過敏 3 1.4血清素受體的分類與路徑 4 1.5與痛覺敏感相關的血清素受體 4 1.5.1血清素受體1A 4 1.5.2血清素受體2A 5 1.5.3血清素受體3A 5 1.5.4血清素受體4 5 1.5.5血清素受體7 6 1.6與機械性痛覺過敏相關的離子通道 6 1.6.1酸敏感離子通道家族 (acid-sensing ion channels, ASICs) 6 1.6.2辣椒素受體1 (transient receptor potential cation channel, subfamily V, member 1, TRPV1) 7 1.6.3電壓調控鈉離子通道 (Voltage-gated sodium channels, Nav ) 7 1.7研究動機與目的 8 第二章 材料與方法 9 2.1實驗材料 10 2.1.1 菌株 10 2.1.2 細胞株 10 2.1.3 實驗動物 10 2.1.4 藥品 10 2.2 實驗方法 11 2.2.1 膠體萃取法 (gel extraction) 11 2.2.2瓊脂膠體的製備及電泳 11 2.2.3聚合酵素連鎖反應 (Polymerase chain reaction, PCR) 11 2.2.4 大腸桿菌的轉型作用(Transformation) 12 2.2.5 細菌培養 12 2.2.6 質體製備 13 2.2.7 細胞培養 14 2.2.8轉染作用(Transfect) 14 2.2.9 鈣離子分析(Calcium imaging) 15 2.2.10動物疼痛行為實驗 16 2.2.11 統計分析 17 第三章 結果 18 3.1 血清素受體2B會受到血清素或血清素受體2促進劑的活化,並且被血清素受體2B/2C的抑制劑抑制 19 3.2 血清素受體2A會受到血清素及血清素受體2促進劑的活化,並且被血清素受體2A的抑制劑抑制 19 3.3 血清素受體2C會受到血清素的活化,並且被血清素受體2B/2C的抑制劑抑制 20 3.4 血清素受體1A會受到血清素的活化,並且被血清素受體1A的抑制劑抑制 20 3.5 血清素受體3A會受到血清素的活化,並且被血清素受體3A的抑制劑抑制 20 3.6 血清素受體2B/2C的抑制劑可以抑制由血清素所引發的機械性痛覺異常,但不能抑制血清素引起的腳腫脹 21 3.7血清素受體2B/2C的抑制劑可以抑制由血清素受體2促進劑所引發的機械性痛覺異常 21 3.8 抑制血清素受體1A和3A的活性不能抑制由血清素所引發的機械性痛覺過敏 22 3.9血清素受體2B不會抑制由血清素引起的熱痛覺過敏 23 3.10 血清素受體2B/2C的抑制劑不能抑制由發炎物質所引發的機械性痛覺過敏 23 3.11 血清素受體2B/2C的抑制劑不能抑制由辣椒素和血清素加pH5.0生理緩衝混合液所引發的機械性痛覺過敏 24 3.12 抑制鈉離子通道可以抑制機械性痛覺過敏的產生 24 3.13 轉染辣椒素受體1,酸敏感離子通道1和3至人類胚胎腎臟細胞大量表現後,受不同濃度辣椒素和不同pH值的酸性溶液刺激後所產生的反應 25 3.14在共同轉染血清素受體2B和辣椒素受體1的細胞中,血清素受體2B的活化會增強辣椒素受體1對鈣離子通透性 25 3.15在共同轉染血清素受體2B和酸敏感離子通道1a和3的細胞中,血清素受體2B的活化會減少酸敏感受體1a和3對鈣離子通透性 26 第四章 討論 28 4.1血清素受體2B參與血清素引起的機械性痛覺過敏 29 4.2血清素受體2B為主要調控血清素引起的痛覺過敏 29 4.3 血清素受體3引起短暫的疼痛,並影響痛覺過敏的維持 30 4.4血清素受體1A對血清素引起的機械性痛覺過敏有些微抵抗疼痛的效果 31 4.5 血清素受體2B/2C不參與辣椒素和血清素加pH5.0酸性生理緩衝液所引發的機械性痛覺過敏 31 4.6血清素受體2B/2C不會調控由發炎物質所引起的機械性痛覺過敏 31 4.7抑制鈉離子通道可抑制機械性痛覺過敏的產生 32 4.8血清素受體引發痛覺過敏的機制 32 第五章 參考文獻 34 附錄 72 圖目錄 圖3.1 血清素受體1A、 2A、 2B、 2C、 3A和4基因圖譜 41 圖3.2 血清素受體2B受到促進劑和抑制劑刺激後調控細胞內鈣離子濃度 43 圖3.3 血清素受體2A受到促進劑和抑制劑刺激後調控細胞內鈣離子濃度 45 圖3.4 血清素受體2C受到促進劑和抑制劑刺激後調控細胞內鈣離子濃度 47 圖3.5 血清素受體1A受到促進劑和抑制劑刺激後調控細胞內鈣離子濃度 49 圖3.6 血清素受體3A受到促進劑和抑制劑刺激後調控細胞內鈣離子濃度 50 圖3.7 血清素受體2B/2C的抑制劑可以抑制由血清素所引發的機械性痛覺異常,但血清素受體2A的抑制劑不能 52 圖3.8 血清素受體2B/2C的抑制劑可以抑制由血清素2促進劑所引發的機械性痛覺異常,但血清素受體2A的抑制劑不能 54 圖3.9 血清素受體1A和3A的抑制劑不能抑制由血清素所引發的機械性痛覺異 56 圖3.10 血清素受體2A的抑制劑可以抑制由血清素所引發的熱痛覺過敏 58 圖3.11 血清素受體2B/2C的抑制劑不能抑制由CFA和carrageenan所引發的機械性痛覺異常 60 圖3.12 血清素受體2B/2C的抑制劑不能抑制由血清素與酸/辣椒素所引發的機械性痛覺異常 62 圖3.13 鈉離子通道的抑制劑能抑制由血清素所引發的機械性痛覺異常 64 圖3.14在人類胚胎腎臟細胞中各種基因的分布 65 圖 3.15 在酸敏感離子受體1a、3和辣椒素受體1基因大量表現的人類腎臟胚胎細胞中,鈣離子變化量和血清素濃度的關係圖 66 圖3.16 血清素受體2B能增強辣椒素受體1的功能 69 圖3.17 血清素受體2B抑制酸敏感離子通道1a和3的功能 71

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