血清素受體2B基因在酸敏感受體3基因剔除小鼠的背根神經節中表現量增加

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黃淳瑩(Chun-Ying Huang) 查詢紙本館藏

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血清素受體2B基因在酸敏感受體3基因剔除小鼠的背根神經節中表現量增加
(Enhanced transcription of serotonin receptor 2B in dorsal root ganglion from ASIC3 knockout mice)

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摘要(中)

在組織受到傷害或者發生發炎反應時，通常會造成疼痛感覺，同時組織四周圍會有組織酸化的現象，組織酸化被認為是造成疼痛的主要原因。在已知可接受酸刺激的受體中，酸敏感受體3被認為是最主要負責接受酸而引發疼痛的受體，然而令人驚訝的是，酸敏感受體3基因剔除小鼠對於一些中高強度刺激所引起的疼痛更敏感，這似乎代表了有另外一些分子彌補了酸敏感受體3的缺失，使得基因剔除小鼠對高強度有害刺激變得更敏感。在實驗室先前的G蛋白偶合受體微陣列分析中，我們發現血清素受體2B是所有血清素受體中唯一表現量上升的基因。然而血清素受體2B在痛覺上的功能及它和酸敏感受體3基因之間的相互作用仍然未知，本篇論文主要的目的是想了解血清素受體2B和酸敏感受體3之間的相互作用。首先，我先確認微陣列分析的結果是否正確，即血清素受體2B是否在酸敏感受體3基因剔除小鼠的背根神經節中，有表現量上升的情形，此外我也要觀察受體2B在背根神經節中的分佈情形。實驗結果發現血清素受體2B表現在所有的組織中，並且在酸敏感3基因剔除小鼠的背根神經節中表現量增加2倍。利用原位雜合反應實驗，我發現血清素受體2B主要分佈在背根神經節中直徑較小的神經元中，而這些直徑較小神經纖維主要負責痛覺訊息傳遞，血清素受體2B不但表現在酸敏感受體3基因剔除小鼠的背根神經節中直徑較小的神經元，且分佈細胞數目有增加的情形，這些結果顯示血清素受體2B可能與痛覺訊息傳遞或調控有關。因為血清素可以增加酸敏感受體3的基因表現，同時在酸敏感受體3基因剔除後血清素受體2B表現量增加，所以我們推測血清素可能藉著受體2B而使酸敏感受體3基因表現增加。為了驗證這個假設，我利用聚合酶鏈反應擴增小鼠的酸敏感受體3啟動子的基因，將此基因選殖入載體用以進行啟動子活性分析。結果發現即使細胞中轉染的血清素受體2B具有活性，血清素亦不能增加酸敏感受體3啟動子的活性。但是，這並不代表在真正生物體中，血清素不能藉由血清素受體2B去影響酸敏感受體3的基因表現，其可能原因我們將於此論文中討論。

摘要(英)

Tissue acidosis, an important feature of tissue injury or inflammation, is a dominant factor causing pain. Among those acid sensing receptors, acid sensing ion channel 3 (ASIC3) is believed to be the major receptor responsible for acid-induced pain. Surprisingly, ASIC3-deficient mice have displayed a higher sensitivity in pain sensation than wild-type mice. It is likely that other molecules compensate ASIC3-deficiency. Using microarray analysis, previous studies have found that the transcripts of serotonin receptor 2B (5-HT2B) are up-regulated in ASIC3 knockout mice. However, the role of 5-HT2B in pain sensation and its relationship with ASIC3 remain unclear. The objective of this thesis is to explore the function of 5-HT2B in pain and to understand the relationship between 5-HT2B and ASIC3. To achieve this aim, I first confirmed the results of microarray and examined 5-HT2B distribuction. 5-HT2B is expressed in all tested tissues from wild-type and ASIC3 knockout mice. The expression is enhanced two folds in dorsal root ganglion (DRG) from ASIC3 knockout mice. Using in situ hybridization, I have found that the majority of 5-HT2B is expressed in a subset of pain-relevant, small-diameter neurons, and this population increases in ASIC3 knockout DRG. Since serotonin increases ASIC3 transcripts and 5-HT2B transcription is enhanced in a loss of ASIC3 gene, it is possible that serotonin enhances ASIC3 transcription through 5-HT2B. To test this hypothesis, I have cloned ASIC3 promoter to examine the promoter activity in the presence of 5-HT2B. After serotonin stimulation, ASIC3 promoter activity is not enhanced, even though 5-HT2B is activated. The possible reasons will be discussed in the thesis.

關鍵字(中)

★ 背根神經節
★ 基因剔除小鼠
★ 啟動子
★ 酸敏感受體3
★ 血清素
★ 血清素受體2B
★ 直徑較小神經元
★ 痛覺

關鍵字(英)

★ nociception
★ dorsal root ganglion
★ DRG
★ knockout mice
★ promoter
★ ASIC3
★ 5-HT2B
★ serotonin receptor 2B
★ serotonin
★ peripherin positive cells
★ small diameter cells
★ pain

論文目次

中文摘要………………………………………………Ⅰ
英文摘要………………………………………………Ⅱ
目錄……………………………………………………Ⅲ
圖目錄…………………………………………………Ⅷ
表目錄…………………………………………………Ⅹ
縮寫與全名對照表……………………………………ⅩI
第一章緒論
1.1痛覺………………………………………………………1
1.2痛覺的訊息傳遞…………………………………………3
1.3發炎反應…………………………………………………4
1.3.1組織酸化………………………………………………………4
1.3.2酸敏感受體家族………………………………………………4
1.3.3 辣椒素受體……………………………………………………6
1.4 血清素及其受體…………………………………………6
1.4.1 血清素及其受體………………………………………………6
1.4.2 訊息傳遞機制…………………………………………………7
1.4.3 血清素受體2B的功能………………………………………8
1.5 動機及目的………………………………………………9
第二章材料與方法
2.1 實驗材料
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
2.1.4.1 購自sigma藥廠………………………………………………………10
2.1.4.2 購自invitrogen公司…………………………………………………10
2.1.4.3 購自Merck藥廠………………………………………………………10
2.1.4.4 購自J.T.Baker藥廠…………………………………………………10
2.1.4.5 購自Fluka公司………………………………………………………11
2.1.4.6 購自Roche公司………………………………………………………11
2.1.4.7 購自Qiagen……………………………………………………………11
2.1.4.8 其他……………………………………………………………………11
2.2 實驗方法
2.2.1 RNA的萃取……………………………………………………12
2.2.1.1 使用TRIzol reagent的方法..............................12
2.2.1.2 使用RNeasy Mini kit的方法………………………………………12
2.2.1.3 染色體DNA(genomic DNA)污染的檢測及處理……………………13
2.2.2 cDNA的合成…………………………………………………13
2.2.3 引子(primer)的設計………………………………………14
2.2.4 聚合酶鏈反應………………………………………………14
2.2.4.1反轉錄-聚合酶鏈反應………………………………………………14
2.2.4.2聚合酶鏈反應………………………………………………………15
2.2.4.3 菌落篩選型聚合酶鏈反應(Colony PCR)…………………………15
2.2.4.4 定量聚合酶鏈反應(Quantitative PCR，Q-PCR)…………………15
2.2.5 聚合酶鏈反應產物的萃取與純化…………………………16
2.2.5.1 瓊脂醣膠的製備及膠體電泳………………………………………16
2.2.5.2 聚合酶鏈反應的產物純化(gel extraction)……………………16
2.2.6 大腸桿菌的轉型作用………………………………………17
2.2.6.1 勝任細胞的備製……………………………………………………17
2.2.6.2 轉型作用……………………………………………………………17
2.2.7 細菌培養……………………………………………………17
2.2.7.1 固體培養……………………………………………………………17
2.2.7.1.1含有Ampicillin的LB培養盤之配製……………………………17
2.2.7.1.2 細菌培養…………………………………………………………18
2.2.7.2 液體培養……………………………………………………………18
2.3.7.3 菌種保存……………………………………………………………18
2.2.8 質體製備……………………………………………………18
2.2.8.1 小量製備的方法(miniprep)………………………………………18
2.2.8.2 大量製備的方法(midiprep)………………………………………18
2.2.9 基因轉殖…………………………………………………… 19
2.2.9.1 載體的製備…………………………………………………………19
2.2.9.2 接合作用(ligation)………………………………………………19
2.2.10 原位雜合反應………………………………………………20
2.2.10.1 探針的備製……………………………………………………… 20
2.2.10.1.1 重組質體pBSII-5HT2B的線性化作用…………………………20
2.2.10.1.2 重組質體的純化……………………………………………… 20
2.2.10.1.3 試管內轉錄作用(in vitro transcription)…………………21
2.2.10.1.4 純化RNA…………………………………………………………21
2.2.10.1.5 RNA定量…………………………………………………………21
2.2.10.2 組織切片…………………………………………………………21
2.2.10.2.1 玻片處裡………………………………………………………21
2.2.11.2.2 組織切片的製作………………………………………………22
2.2.10.2.3 組織切片的固定和乙烯化……………………………………22
2.2.10.3 RNA雜合反應及呈色反應…………………………………………22
2.2.10.3.1 雜合反應(hybridization)……………………………………22
2.2.10.3.2 免疫染色………………………………………………………23
2.2.11 細胞培養……………………………………………………23
2.2.12 轉染作用……………………………………………………24
2.2.12.1 玻片的前處理……………………………………………………24
2.2.12.2 轉染作用…………………………………………………………24
2.2.13 鈣離子分析…………………………………………………24
2.2.14 促進子活性分析……………………………………………26
2.2.14.1 構築質體（pGL3-mASIC3 promoter）……………………………26
2.2.14.2細胞轉染與施打藥劑………………………………………………26
2.2.14.3促進子活性分析……………………………………………………26
2.2.15發炎反應實驗………………………………………………27
第三章結果
3.1在酸敏感受體3基因剔除小鼠的背根神經節中，血清素受體2B基因轉錄增加…………………………………28
3.2 血清素受體2B在背根神經節細胞的分布情………30
3.3 血清素受體2B與酸敏感受體3的促進………………31
3.4 酸敏感受體3的促進子之序列分析……………………33
3.5 血清素受體2亞族的組織分布與基因序列分析………34
3.6 不同形式的血清素受體2B ……………………………35
3.7 在發炎反應中血清素受體2B與酸敏感受體3在背根神經節上的表現量變化 …………………………………36
第四章討論 ……………………………………37
第五章參考文獻 ………………………………42
附錄一……………………………………………70
附錄二……………………………………………72

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指導教授

孫維欣(Wei-hsin Sun)

審核日期

2005-12-20

推文