MrgB4受體專一表現於感覺神經元，且在ASIC3基因剔除小鼠中有不同的表現。

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張晉毓(Chin-yu Chang) 查詢紙本館藏

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論文名稱

MrgB4受體專一表現於感覺神經元，且在ASIC3基因剔除小鼠中有不同的表現。
(Expression of a sensory neuron-specific receptor, mrgB4,is changed in ASIC3 knockout mice.)

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摘要(中)

Mas1相關基因家族為一G蛋白偶合受體家族，目前發現能專一表現於哺乳類痛覺相關感覺神經元。Mas1相關基因家族受體能被一些特定的內生性氨基化合物神經胜肽活化，例如：神經胜肽FF能活化小鼠的Mas1相關基因受體A1及C11、神經胜肽AF能活化小鼠的Mas1相關基因受體A4、γ2-促黑激素能活化大鼠的Mas1相關基因受體C、以及牛腎上腺髓肽22能活化人類的Mas1相關基因受體X1，而且不同胜肽對於不同家族受體之間的活化程度存在著相異性。一些Mas1相關基因受體在活化後能抑制特定調控痛覺的陽離子通道，如：鉀離子、鈉離子、或鈣離子通道。不過，Mas1相關基因B亞家族是否表現於痛覺感覺神經元，以及受體是否受專一的氨基化合物胜肽活化，均尚未了解。因此，此篇論文的目標在於偵測Mas1相關基因B亞家族其組織表現性及確認可能活化物。在反轉錄-聚合酵素連鎖反應後發現只有Mas1相關基因B4及B5能表現在背根神經節，但所偵測的Mas1相關基因B亞家族均表現於三叉神經節。另外感到有趣的地方，Mas1相關基因B4轉錄作用在酸敏感受體3剔除小鼠有顯著地變化，而經由原位雜合反應確定這些結果。這些結果暗示著Mas1相關基因B4的功能在與小鼠酸敏感受體3所引起的痛覺敏感有關。使用氨基化合物胜肽刺激於表現Mas1相關基因B4受體的人類腎臟胚胎細胞上，Mas1相關基因B4受體在被γ2-促黑激素活化後，利用Gi蛋白傳遞胞內訊息。然而，γ2-促黑激素必須使用過量的濃度才能活化Mas1相關基因B4受體，因此，並未確定出Mas1相關基因B4受體之專一活化物。

摘要(英)

The subfamily B of Mas1-related gene (MrgBs) belongs to a family of G-protein coupled receptors (GPCRs) that are recently identified in mammals and specifically expressed in primary nociceptive (pain-related) sensory neurons. Reflecting the diversity and specificity of stimuli they respond, Mrgs are activated by different peptides containing RFamide motif, such as NPFF for mouse MrgA1 and C11, NPAF for mouse MrgA4, γ2-MSH for rat MrgC/SNSR1, and BAM22 for human MrgX1/SNSR3 and SNSR4. Activations of some Mrg receptors can inhibit the specific potassium and calcium channels that modulate nociception. However, whether MrgBs are involved in nociception and whether they are actived by RFamide peptides remain unknown. Therefore, the aim of this thesis is to examine tissue distribution of MrgB genes and identify their ligands. In all of MrgB members tested, only MrgB4 and B5 transcripts were expressed in dorsal root ganglion (DRG), but all MrgB genes were expressed in trigeminal ganglion (TG). Interestingly, MrgB4 transcripts were significantly changed in acid-sensing ion channel 3 (ASIC3) deficient mice and such results were confirmed using in situ hybridization. These results suggest that MrgB4 may have functions related to the enhanced sensitivity of ASIC3+/+ mice to painful stimuli. Using RFamide pepides to test MrgB4-overexpressing HEK293 cells, MrgB4 was activated by γ2-MSH through the Gi pathway. Nevertheless, the concentration of γ2-MSH for MrgB activation was too high, the specific ligands for MrgB4, therefore, are inconclusive.

關鍵字(中)

★ 感覺神經元
★ 三叉神經節
★ 背根神經節
★ 酸敏感受體3

關鍵字(英)

★ mrgB4
★ ASIC3
★ DRG
★ TG

論文目次

中文摘要 i
英文摘要 ii
致謝 iii
目錄 iv
圖目錄 ix
表目錄 xi
縮寫與全名對照表......xii
第一章緒論 1
1.1痛覺 2
1.1.1 痛覺神經元 (Nociceptors) 2
1.1.2 痛覺的訊息傳遞 3
1.2 發炎反應 4
1.2.1 組織酸化 4
1.2.2 酸敏感受體家族及辣椒素受體 4
1.2.2.1 酸敏感受體家族 4
1.2.2.2 辣椒素受體 5
1.3 鴉片家族 (Opioid Family) 6
1.3.1 腦啡肽前質 (proenkephalin) 6
1.3.2 強啡肽前質 (prodynorphin) 6
1.3.3 孤啡肽前質 (proorphanin) 7
1.3.4 原嗎啡黑色素皮質素 (proopiomelanocortin，POMC) 7
1.4 神經肽FF (Neuropeptide FF) 8
1.5 Mas1相關基因家族(Mrg，Mas1-related gene) 8
1.5.1 Mas1相關基因家族表現於感覺神經細胞 9
1.5.2 Mas1相關基因家族受體之活化 9
1.5.3 Mas1相關基因家族受體之生理功能 10
1.6 動機及目的 11
第二章材料與方法 12
2.1 表現質體的建立與製備 13
2.1.1 DNA片段的製備 13
2.1.1.1 質體之限制酶切割 13
2.1.1.2 瓊脂醣膠的製備及電泳 13
2.1.1.3 DNA片段之純化 13
2.1.2 載體的製備 14
2.1.3 接合作用 14
2.1.4 大腸桿菌的轉型作用 (Transformation) 14
2.1.5 表現質體的製備 14
2.1.5.1 小量的製備 (miniprep) 14
2.1.5.2 限制酶分析 15
2.1.5.3 大量的製備 (midiprep) 15
2.2 聚合酵素連鎖反應 (Polymerase chain reaction, PCR) 16
2.2.1 引子 (primer) 的設計 16
2.2.2 各個組織cDNA的合成 16
2.2.2.1 組織的取得 16
2.2.2.2 核醣核苷酸(RNA)的萃取 17
2.2.2.2.1 使用RNeasy kit方法 17
2.2.2.2.2 使用TRIzol reagent方法 17
2.2.2.3 cDNA的合成 18
2.2.3 反轉錄-聚合酵素連鎖反應 (RT-PCR) 18
2.2.4 定量聚合酵素連鎖反應 (Quantitative PCR, Q-PCR) 19
2.3 原位雜合反應 (In situ hybridization) 與免疫染色
(Immunohistochemistry) 19
2.3.1 探針的製備 20
2.3.1.1 重組質體的線性化 (Linear) 與純化 20
2.3.1.2 試管內轉錄作用 (In vitro tanscription) 20
2.3.1.3 RNA探針的純化 20
2.3.1.4 RNA探針的定量 21
2.3.2 組織切片的製作 21
2.3.2.1 玻片的處理 21
2.3.2.2 組織切片的固定、及乙烯化 21
2.3.3 原位雜合反應 (In situ hybridization) 22
2.3.4 免疫染色 (Immunohistochemistry) 23
2.4 細胞培養 (cell culture) 與轉染作用 (transfection) 23
2.4.1細胞培養 (cell culture) 23
2.4.2 轉染作用 24
2.4.2.1 玻片的處理 24
2.4.2.2 轉染作用 24
2.5 白日咳毒素 (Pertussis toxin，PTX) 的刺激 24
2.6 細胞內鈣離子濃度的分析 25
2.7 發炎實驗 26
第三章結果 27
3.1 小鼠的Mas1相關基因B亞家族於胺基酸序列上有著高度相似的
程度。28
3.2 小鼠的Mas1相關基因B亞家族於野生型及酸敏感受體 3基因剔除
小鼠各個組織中有著不同的表現。 28
3.3 小鼠的Mas1相關基因B亞家族之表現程度在野生型及酸敏感受體
3基因剔除小鼠背根神經節與三叉神經節之間的比較。29
3.4 小鼠的Mas1相關基因B4於野生型小鼠內個別腰椎背根神經節中有著不同的表現量。30
3.5 小鼠的Mas1相關基因B4於野生型及酸敏感受體3基因剔除小鼠背
根神經節中，在小、中、和大直徑細胞的分佈情況。30
3.6 於野生型及酸敏感受體3基因剔除小鼠背根神經節中，IB4+ 和
IB4-細胞其表現小鼠的Mas1相關基因B4之比例多寡。31
3.7 於野生型及酸敏感受體3基因剔除小鼠背根神經節中，小鼠的
Mas1相關基因B4在具或不具辣椒素受體之神經細胞內其表現
情況。 32
3.8 小鼠的Mas1相關基因B4於野生型及酸敏感受體3基因剔除小鼠
三叉神經節中，在小、中、和大直徑細胞的分佈情況。33
3.9 於野生型及酸敏感受體3基因剔除小鼠三叉神經節中，IB4+和
IB4- 細胞其表現小鼠的Mas1相關基因B4之比例多寡。34
3.10 於野生型及酸敏感受體3基因剔除小鼠三叉神經節中，小鼠的
Mas1相關基因B4在具或不具辣椒素受體之神經細胞內其表現
情況。 34
3.11 三種內生性神經胜肽 (神經肽FF、γ2-促黑激素、及牛腎上腺
髓肽22) 的刺激，造成小鼠的Mas1相關基因B4受體對於細胞
內鈣離子濃度改變之程度。 35
3.12 在發炎反應中，小鼠的Mas1相關基因B4於野生型小鼠背根神
經節中表現無明顯的變化。37
第四章討論 38
4.1 小鼠的Mas1相關基因B4能專一表現於感覺神經組織。 39
4.2 小鼠的Mas1相關基因B4於酸敏感受體3基因剔除小鼠感覺神經組
織中有著不同的表現，不過在發炎反應中，於背根神經節中其表現量並無明顯的變化。41
4.3 小鼠的Mas1相關基因B4於個別腰椎背根神經節中有著不同的表現量，以及於背根神經節及三叉神經節中，在不同類型神經細胞的分
佈情況。41
4.4 小鼠的Mas1相關基因B4受體之可能活化物，以及活化後於細胞內所參與的機制。 43
第五章參考文獻 46
附錄 102

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指導教授

孫維欣(Wei-hsin Sun)

審核日期

2007-7-20

推文