酸敏感G蛋白偶合受體,G2A,在ASIC3基因剔除小鼠中改變表現量

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蔡維棻(Wei-Fen Tsai) 查詢紙本館藏

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論文名稱

酸敏感G蛋白偶合受體,G2A,在ASIC3基因剔除小鼠中改變表現量
(Expression change of proton-sensing G-protein coupled receptor,G2A,in ASIC3 knockout mice)

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摘要(中)

組織的受損與發炎，往往引起局部組織中氫離子濃度的增加，造成組織酸化。組織酸化被認為是導致疼痛的主要原因，目前已知有兩個陽離子通道，酸敏感離子通道家族(acid-sensing ion channel；ASIC3)及辣椒素受體（vanilloid receptor 1；VR1），可以受到氫離子的活化，並且參與傷害感受覺（nociception）的傳遞。最近的研究發現，屬於酸敏感G 蛋白偶合受體（proton-sensing G-protein-coupledreceptors）的OGR1 接受體家族，也可以接收氫離子的刺激，包括了：OGR1、GPR4 、TDAG8 及G2A。G2A 在早期認為是LPC（脫脂磷酸脂膽鹼，lysophosphatidylcholine ）的接受體，並且受到氫離子及9-HODE（9-hydroxyoctadecadienoic）的活化。然而，G2A 是否可被氫離子所活化，並且是否參與傷害感受覺的調控機制，仍然不清楚。本篇論文的主要目的，是在探討氫離子對於G2A 的影響以及表現形式。經由RT-PCR 的結果顯示，G2A 在許多組織中均有表現，包括了背根神經節（dorsal root ganglia；DRG）。G2A 主要表現在小直徑，IB4-positive 的傷害感受器（nociceptor）。其中有趣的是，G2A 增加表現量在ASIC3 基因剔除小鼠DRG 中，並且主要增加在大直徑細胞。因此，G2A 也許參與了傷害感受覺的調控。然而在氫離子對G2A 的影響方面，與前人的實驗結果相符，我發現G2A 無法被氫離子所活化。

摘要(英)

Tissue injury and inflammation often cause an increase of hydrogen ion concentration in local tissues, called tissue acidosis. Tissue acidosis seems to be the dominant factor that leads to painful sensation. Two cation channels,acid-sensing ion channel(ASIC3) and vanilloid receptor 1, are activated by proton and involved in nociceptive transduction. Recently, a subfamily of G-protein-coupled receptor (GPCR) including OGR1, GPR4, TDAG8 and G2A has been identified as proton-sensing receptors. G2A is originally known as a lysophosphatidylcholine (LPC) receptor and is also actived by proton
and 9-hydroxyoctadecadienoic. However, original studies for LPC and proton cannot be reproducible. Whether G2A is activated by proton and whether it is involved in nociception remain unclear. The objective of this thesis is to determine effects of pH on G2A and study its expression pattern. From RT-PCR results, G2A was expressed in many tissues including dorsal root ganglia (DRG). G2A was predominantly expressed in small-diameter, IB4-positive nociceptors. Interestingly, expression levels of G2A increased in ASIC3-/-DRG. This increase is due to an increase in G2A-expressing neurons, mainly in large diameter neurons. Accordingly, G2A may be involved in nociception. Consistent with previous studies, I have found G2A cannot be activated by proton.

關鍵字(中)

★ G-蛋白偶合受體
★ 痛覺
★ 背根神經節
★ 組織酸化
★ G2A
★ ASIC3

關鍵字(英)

★ tissue acidosis
★ ASIC3
★ GPCR
★ G2A
★ Pain
★ DRG

論文目次

中文摘要…………………………………………………………………………………...Ⅰ
英文摘要…………………………………………………………………………………...Ⅱ
致謝………………………………………………………………………………………...Ⅲ
目錄…………………………………………………………………………………...........Ⅳ
圖目錄………………………………………………………………………………….......Ⅶ
表目錄………………………………………………………………………………….......Ⅶ
縮寫與全名對照表………………………………………………………………………...Ⅷ
第一章序論
1.1 痛覺………………………………………………………………………....................2
1.1.1 傷害感受性受器…………………………………………………………………….3
1.1.2 傷害感受覺的傳遞路徑…………………………………………………………….4
1.1.2.1 脊髓………………………………………………………………………………4
1.1.2.2 傷害感受覺的脊髓傳遞路徑……………………………………………………5
1.1.2.3 由脊髓傳遞到視丘及大腦皮質的路徑………………………………………..5
1.2 發炎反應及組織酸化…………………………………………………………………..6
1.2.1 發炎反應……………………………………………………………………………..7
1.2.2 發炎反應調節物……………………………………………………………………..8
1.2.3 組織酸化…………………………………………………………………………..10
1.3 氫離子接受體………………………………………………………………………….11
1.3.1 辣椒素受體 VR1…………………………………………………………………...11
1.3.2 酸敏感離子通道家族………………………………………………………………12
1.3.3 酸敏感G 蛋白偶合受體……………………………………………………………13
1.3.3.1 OGR1………………………………………………………………………........14
1.3.3.2 GPR4……………………………………………………………………….........14
1.3.3.3 TDAG8…………………………………………………………………………..15
1.3.3.4 G2A……………………………………………………………………………...15
1.4 研究目的………………………………………………………………………………17
第二章實驗材料及方法
2.1 RNA 的萃取與備製……………………………………………………………………19
2.1.1 小鼠組織的製備……………………………………………………………………19
2.1.2 大量RNA 萃取……………………………………………………………………..19
2.1.3 小量RNA 萃取……………………………………………………………………..20
2.1.4 RNA 品質及濃度的測量…………………………………………………………..20
2.1.4.1 瓊脂醣膠(agarose gel)的製備及膠體電泳……………………………………20
III
2.1.4.2 RNA 變性……………………………………………………………………….21
2.1.4.3 RNA 濃度的測量……………………………………………………………….21
2.1.5 染色體DNA 的污染檢測及處理…………………………………………………..21
2.1.5.1 染色體DNA 的汙染檢測………………………………………………………21
2.1.5.3 DNase I 的處理...................................................................................................22
2.1.5.4 cDNA 的合成.....................................................................................................22
2.2 聚合酶連鎖反應.............................................................................................................22
2.2.1 引子的設計...............................................................................................................22
2.2.2 複製GPCR 基因的聚合酶連鎖反應.....................................................................23
2.2.3 反轉錄聚合酶連鎖反應.........................................................................................23
2.2.4 定量聚合酶連鎖反應.............................................................................................24
2.3 mG2A 基因的複製及質體的製備.................................................................................24
2.3.1 載體的製備...............................................................................................................24
2.3.1 mG2A 基因的複製...................................................................................................25
2.3.2 膠體萃取...................................................................................................................25
2.3.3 接合作用...................................................................................................................25
2.3.4 轉染作用...................................................................................................................26
2.3.5 PCR 菌落篩選..........................................................................................................26
2.3.6 小量質體製備...........................................................................................................26
2.3.7 大量質體製備...........................................................................................................27
2.3.8 mG2A 基因載體的重新構築...................................................................................27
2.4 原位雜合反應.................................................................................................................27
2.4.1 探針的製作..............................................................................................................28
2.4.1.1 製備mG2A基因反意義股探針的模板.............................................................28
2.4.1.2 mG2A 基因反意義股探針模板的純化.............................................................28
2.4.1.3 mG2A 基因反意義股探針的製備......................................................................28
2.4.1.4 mG2A 基因探針的純化.......................................................................................29
2.4.2 組織切片的製備....................................................................................................29
2.4.2.1 玻片處理..............................................................................................................29
2.4.2.2 組織切片..............................................................................................................29
2.4.2.3 組織切片的固定及乙醯化..................................................................................30
2.4.2.4 雜合反應..............................................................................................................30
2.4.4.5 免疫染色...............................................................................................................31
2.5 鈣離子分析實驗............................................................................................................32
2.5.1 細胞培養..................................................................................................................32
2.5.1.1 人類胚胎腎臟細胞.............................................................................................32
2.5.1.2 N2A....................................................................................................................32
IV
2.5.2 轉染作用................................................................................................................33
2.5.2.1 玻片前處理........................................................................................................33
2.5.2.2 種細胞.................................................................................................................33
2.5.2.3 細胞轉染.............................................................................................................33
2.5.3 鈣離子分析實驗.....................................................................................................34
2.5.3.1 pH 緩衝溶液的配製...........................................................................................34
2.5.3.2 Fura-2 的前處理.................................................................................................34
2.5.3.3 給予不同pH值刺激的鈣離子分析...................................................................34
第三章結果
3.1 OGR1 基因家族在ASIC3+/+及ASIC3-/-小鼠在各組織中的分佈情形........................35
3.2 mG2A基因的表現量增加在ASIC3 -/-小鼠的背根神經節中........................................35
3.3 mG2A基因的表現量增加在ASIC3 -/-小鼠背根神經節的大細胞中...........................35
3.4 mG2A基因轉移表現量在ASIC3 -/-小鼠背根神經節IB4-的小細胞中........................36
3.5 mG2A基因在ASIC3 -/-小鼠背根神經節的表現改變至VR1-的小細胞中.................37
3.6 在過表現G2A 基因的細胞中G2A 基因對於不同pH 值的反應...............................37
第四章討論.........................................................................................................................40
參考文獻...............................................................................................................................69
附錄......................................................................................................................................74

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指導教授

孫維欣(Wei-Hsin Sun)

審核日期

2007-7-22

推文