血清素受體2B對酸敏感離子通道3與辣椒素受體1的影響

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林志信(Chih-hsin Lin) 查詢紙本館藏

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論文名稱

血清素受體2B對酸敏感離子通道3與辣椒素受體1的影響
(The effects of serotonin receptor 2B on the acid-sensing ion channel 3 (ASIC3) and transient receptor potential cation channel 1(TRPV1))

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摘要(中)

當組織受傷而產生發炎反應時，會有發炎物質從細胞中釋出，以及組織局部酸化的現象。造成組織酸化的高濃度氫離子會活化神經元上的一些離子通道，如酸敏感受體3(ASIC3)、辣椒素受體1(TRPV1)等，造成發炎痛與痛覺過敏的現象，而同時由細胞中釋出的發炎物質會加強痛覺過敏的現象。血清素是一種發炎物質，現今已知可以調節大鼠酸敏感離子通道3基因表現與辣椒素受體1離子通道反應，可是血清素是藉由哪個受體及機制來發揮它的作用仍然不是很清楚，本篇論文的主題就是在探討在小鼠發炎中酸敏感離子通道3與辣椒素受體1的變化以及血清素能否藉由血清素受體2B調節一些離子通道的特性。我們發現當注射CFA引起小鼠發炎時，小鼠背根神經上的酸敏感受體3與辣椒素受體1的基因表現量並沒有改變，而血清素受體2亞族表現量有增加的現象。而在血清素注射到小鼠中或直接刺激小鼠體外培養感覺神經元，酸敏感離子通道3與辣椒素受體1基因表現都沒有明顯改變，推測發炎時痛覺傳遞調控不是經由影響酸敏感離子通道3與辣椒素受體1基因表現造成。之前文獻指出血清素能增強辣椒素受體1離子通道的反應，並可受血清素受體2與4亞族抑制劑抑制，我們將血清素受體2B與辣椒素受體1共同轉染到人類胚胎腎臟細胞中，在依序刺激血清素與辣椒素，發覺血清素刺激血清素受體2B後辣椒素受體對於辣椒素的反應很明顯增強，推測血清素可經由血清素受體2B來增強辣椒素受體1的反應。

摘要(英)

Inflammation induced by tissue injury, infection or tumor growth often produces chronic and persistent pain. A key factor of inflammation is tissue acidosis that contributes directly in pain and hyperalgesia. This acid-evoked pain is potentiated by inflammatory mediators released from the primary sensory terminal and from non-neural cells in the environment. It has been proven that the transcripts of acid-sensing ion channel 3(ASIC3) and the transient receptor potential V1(TRPV1) are enhanced during nflammation or after stimulation of inflammatory mediators such as serotonin(5-hydroxytryptamine, 5-HT), bradykinin, nerve growth factor. The channel excitability of ASIC3 and TRPV1 is also enhanced by inflammatory mediators. However, it remains unclear how serotonin regulate sASIC3 and TRPV1 functions in sensory neuron. The objective of this thesis is to identify the effect of serotonin receptors on ASIC3 and TRPV1. To address that, gene expression of ASIC3 and TRPV1 was first examined after complete Freund’s adjuvant-induced inflammation or serotonin injection, and then signaling of serotonin receptors 2B and TRPV1 was characterized. Unexpectedly, gene expression of mouse ASIC3 and TRPV1 was not changed after inflammation or serotonin injection. But HTR2B activation could potentiate TRPV1 response to capsaicin. We suggested that serotonin could enhance TRPV1 response through HTR2B by modulating channel property, not by gene expression.

關鍵字(中)

★ 血清素
★ 血清素受體2B
★ 辣椒素受體1

關鍵字(英)

★ HTR2B
★ serotonin
★ TRPV1

論文目次

中文摘要………………………………………………Ⅰ
英文摘要………………………………………………Ⅱ
目錄……………………………………………………Ⅳ
圖目錄…………………………………………………Ⅹ
表目錄…………………………………………………ⅩII
第一章. 緒論
1-1.痛覺………………………………………………………2
1-1-1.痛覺感受體………………………………………………2
1-1-2.痛覺傳遞…………………………………………………3
1-2.發炎痛……………………………………………………4
1-2-1.組織酸化………………………………………………………4
1-2-2.發炎物質………………………………………………………5
1-3.離子通道…………………………………………………6
1-3-1.辣椒素受體1…………………………………………………6
1-3-2.酸敏感離子通道3……………………………………………7
1-4.血清素與血清素受體…………………………………………8
1-5.動機及目的………………………………………………10
第二章材料與方法
2.1 實驗材料
2.1.1 菌株……………………………………………………………12
2.1.2 細胞株…………………………………………………………12
2.1.3 實驗動物………………………………………………………12
2.1.4 藥品……………………………………………………………12
2.1.4.1 購自sigma藥廠……………………………………………12
2.1.4.2 購自invitrogen公司………………………………………12
2.1.4.3 購自Merck藥廠…………………………………………… 12
2.1.4.4 購自J.T.Baker藥廠…………………………………………12
2.1.4.5 購自Fluka公司………………………………………………12
2.1.4.6 購自Qiagen公司………………………………………… 13
2.1.4.7 其他…………………………………………………………13
2.2 實驗方法
2-2-1.大腸桿菌的轉型作用…………………………………………14
2-2-1-1.勝任細胞的備製……………………………………………14
2-2-2-2.轉型作用……………………………………………………14
2-2-2.細菌培養………………………………………………………14
2-2-2-1.固體培養……………………………………………………14
2-2-2-2.含有Ampicillin的LB培養盤之配製……………………… 14
2-2-2-3.細菌塗盤培養………………………………………………14
2-2-2-4.液體培養………………………………………………………………15
2-2-2-5.菌種保存……………………………………………15
2-2-3.質體製備………………………………………………………15
2-2-3-1.小量製備的方法(miniprep)………………………………15
2-2-3-2.大量製備的方法(midiprep)………………………………16
2-2-4.基因轉殖………………………………………………………17
2-2-4-1.載體的製備…………………………………………………17
2-2-4-2.接合作用(ligation)………………………………………17
2-2-5.RNA的萃取……………………………………………………18
2-2-5-1.使用TRIzol reagent的方法………………………………18
2-2-5-2.使用RNeasy Mini kit的方法…………………………… 18
2-2-5-3.染色體DNA(genomic DNA)污染的檢測及處理……………19
2-2-6.cDNA的合成………………………………………………… 19
2-2-6-1.一般組織cDNA合成…………………………………………19
2-2-6-2.背根神經節與三叉神經節cDNA合成……………………… 19
2-2-7.引子(primer)的設計…………………………………………20
2-2-8.聚合酶鏈反應………………………………………………… 20
2-2-8-1.反轉錄-聚合酶鏈反應………………………………………20
2-2-8-1.定量聚合酶鏈反應(Quantitative PCR，Q-PCR)…………21
2-2-9.聚合酶鏈反應產物的萃取與純化…………………………… 21
2-2-9-1.瓊脂醣膠的製備及膠體電泳……………………………… 21
2-2-9-2.聚合酶鏈反應的產物純化(gel extraction)……………21
2-2-10.細胞培養………………………………………………………22
2-2-11.轉染作用………………………………………………………22
2-2-11-1.玻片的前處理………………………………………………22
2-2-11-2.轉染作用……………………………………………………22
2-2-12.鈣離子分析……………………………………………………23
2-2-13.發炎反應實驗…………………………………………………24
第三章結果
3-1.在注射稀釋CFA引起發炎的小鼠背根神經節中，血清素受體2亞族基因表現量有顯著增加，酸敏感離子通道3與辣椒素受體1則沒有顯著變化。…………………………………………………………26
3-2.在注射CFA引起發炎的小鼠背根神經節中，酸敏感離子通道3基因表現明顯下降，但血清素受體2B沒有顯著變化。………………………………………………………………26
3-3.在carrageenan引起的發炎反應中，酸敏感離子通道3沒明顯變化，血清素受體2B則有增加的現象。……………………………27
3-4.在血清素刺激的小鼠背根神經節中血清素受體2C表現量大量增加。…………………………………………………………… 27
3-5. 將小鼠背根神經節作成的primary culture用血清素刺激後，酸敏感受離子通道3，辣椒素受體1與血清素受體2B基因表現都沒有明顯變化。………………………………………………………… 27
3-6. 血清素受體基因分布在小鼠的背根神經節中。 ……………………………………………………………28
3-7. 血清素受體2B轉染至人類胚胎腎臟細胞大量表現後，受血清素刺激後產生的反應。……………………………………………… 28
3-8. 在人類胚胎腎臟細胞中大量表現的血清素受體2B在不同環境或抑制劑的影響下所產生的反應。………………………………… 29
3-9. 辣椒素受體1轉染至人類胚胎腎臟細胞大量表現後，受辣椒素刺激後產生的反應。……………………………………………… 30
3-10. 在血清素受體2B與辣椒素受體1共同轉染的細胞中，血清素受體的活畫會增強辣椒素受體1活化產生的反應。……………30
第四章討論
4-1.在小鼠注射CFA、carrageenan引起發炎後，酸敏感離子通道3基因表現量無顯著改變。……………………………………………32
4-2.辣椒素受體1在CFA引起發炎後的小鼠背根神經節中基因表現量並沒有明顯改變。…………………………………………………… 33
4-3.血清素受體2亞族在CFA引起的發炎反應基因表現有增加的現象。……………………………………………………………… 34
4-4.在carrageenan引起發炎的小鼠中，酸敏感離子通道3基因沒有明顯改變，但血清素受體2B表現量增加。……………………… 35
4-5.在小鼠背根神經節經血清素刺激後，酸敏感受離子通道3與辣椒素受體1基因都沒有明顯改變。…………………………………… 36
4-6.在小鼠背根神經節經血清素刺激後，血清素受體基因都沒有明顯改變。……………………………………………………………… 36
4-7.血清素受體2B訊息傳遞是經由Gq路徑，轉染到人類腎臟胚胎細胞後會受磷脂酶抑制劑與受體抑制劑抑制。…………………………37
4-8.在血清素受體2B與辣椒素受體1轉染的細胞中，血清素受體2B的活化可以增強辣椒素受體1的反應。………………………………38
第五章參考文獻 ……………………………………………… 71
附錄……………………………………………………………………77

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指導教授

孫維欣(Wei-hsin Sun)

審核日期

2007-7-27

推文