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Student Number 93224008
Author Wei-Fen Tsai(蔡維棻)
Author's Email Address No Public.
Statistics This thesis had been viewed 2397 times. Download 5 times.
Department Life Science
Year 2006
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Expression change of proton-sensing G-protein coupled receptor,G2A,in ASIC3 knockout mice
Date of Defense 2007-07-05
Page Count 89
Keyword
  • ASIC3
  • DRG
  • G2A
  • GPCR
  • Pain
  • tissue acidosis
  • Abstract 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.
    Table of Content 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
    Reference Bevan, S., and Yeast, J. (1992). Protons activate a cation conductance in a
    sub-population of rat dorsal root ganglion neurons. J. Physiol. 433, 145-161.
    Caterina, M.J., Leffler, A., Malmberg, A.B., Martin, W.J., Trafton, J., Petersen-Zeitz, K.R.,
    Koltzenburg, M., Basbaum, A.I. and Julius, D. (2000). Impaired nociception and pain
    sensation in mice lacking the capsaicin receptor. Science 288, 306-313.
    Chen, C.C., Zimmer, A., Sun, W.H., Hall, J., Brownstein, M.J. and Zimmer, A. (2002). A role for
    ASIC3 in the modulation of high-intensity pain stimuli. Proc. Natl. Acad. Sci. U. S. A. 99,
    8992-8997.
    Dirajlal S., Pauers L. E., and Stucky C. L. (2003). Differential responses properties of IB4-positve
    and –negative ummyelinated senspry neurons to protons and capsaicin. J. Neurophysiol. 89,
    513-524.
    Dray, A. Inflammatory mediators of pain. (1995). British Journal of Anaesthesia. 75, 125-131.
    Im, D., Heise, C.E., Nguyen, T., O’Dowd, B.F. and Lynch, K.R. (2001). Identification of a
    molecular target of psychosine and its role in globoid cell formation. J. Cell Biology 153,
    429-434.
    Ishii, Satoshi., Kihara, Yasuyuki., and Takao Shimizu.(2005). Identification of T Cell
    Death-associated Gene 8 (TDAG8) as a Novel Acid Sensing G-protein-coupled Receptor. J. Biol.
    Chem., 280-10, 9083-9087.
    70
    Julius, D. and Basbaum, A.I. (2001). Molecular mechanisms of nociception. Nature 413, 203-210.
    Jones, Nicholas G., Rebeccah Slater., Herve Cadiou., Peter McNaughton., and Stephen B.
    McMahon. (2004). Acid-Induced Pain and Its Modulation in Humans. Progress in Neurobiology,57,
    1-164.
    Kandel, E.R., Schwartz J.H. and Jessell, T.M., Principles of neural science. 4th edition. Chapter 24.
    Kim, Kwan-sik ., Juan Ren., Ying Jiang., Quteba Ebrahem., Russell Tipps., Kelly Cristina., Yi-jin
    Xiao., Jing Qiao.,Kevin L. Taylor.,Hazel Lum., Bela Anand-Apte., and Yan Xu. (2005). GPR4
    plays a critical role in endothelial cell function and mediates the effects of
    sphingosylphosphorylcholine. The FASEB Journal, 1-27.
    Le, Lu Q., Janusz H. S. Kabarowski, Zhigang Weng, Anne B. Satterthwaite, Eric T. Harvill,
    Eric R. Jensen, Jeff F. Miller and Owen N. Witte. (2001). Mice Lacking the Orphan G
    Protein-Coupled Receptor G2A Develop a Late-Onset Autoimmune Syndrome. Immunity 14-
    5, 561-571.
    McCudden,C. R., Hains, M. D., Hains, Kimple., R. J.,Siderovski D. P. and F. S.Willard .(2005).
    G-protein signaling: back to the future. CMLS, Cell. Mol. Life Sci,62 , 551–577.
    McMahon, Stephen B., William B.J. Cafferty., Fabien Marchand. (2005). Immune and glial cell
    factors as pain mediators and modulators. Experimental Neurology 192, 444– 462.
    Millan, Mark J. (1999). The induction of pain: an integrative review. Neurobiology 57- 1, 1-164.
    Molliver, Derek C., Immke , Immke ,David C ., Leonardo, Fierro., Michel, Paré., Frank L Rice2
    and Edwin W McCleskey (2005). ASIC3, an acid-sensing ion channel, is expressed in
    71
    metaboreceptive sensory neurons. Molecular Pain , 1:35, 1-13.
    Murakami, N .,Yokomizo, T., Okuno, T. and Shimizu, T. (2004). G2A is a proton-sensing
    G-protein-coupled receptor antagonized by lysophosphatidylcholine. J. Biol. Chem. 279,
    42484-42491.
    Obinata, Hideru., Tomoyasu Hattori., Shinji Nakane.,Kazuaki Tatei., and Takashi Izumi.
    (2005). Identif cation of 9-Hydroxyoctadecadienoic Acid and Other Oxidized Free Fatty Acids as
    Ligands of the G Protein-coupled Receptor G2A. . Biol. Che., 280-49, 40676-40683.
    Reeh, P. W., and Steen, K. H. (1996). Tissue acidosis in nociception and pain. Brain Research, 113,
    143-151.
    Rikitake, Yoshiyuki., Ken-ichi Hirata., Tomoya Yamashita., Kenji Iwai., Seiichi
    Kobayashi.,, Hiroshi Itoh., Masanori Ozaki., Junya Ejiri., Masashi Shiomi., Nobutaka Inoue.,
    Seinosuke Kawashima., Mitsuhiro Yokoyama. (2002). Arterioscler Thromb Vasc Biol, 22,
    2049-2053.
    Scholz, Joachim and Clifford J. Woolf. (2002). Can we conquer pain? Nature Neuroscience 5,
    1062 – 1067.
    Sluka, Kathleen A., Margaret P. Price., Nicole M. Breese., Cheryl L. Stucky., John A. Wemmie.,
    Michael J. Welsh. (2003). Chronic hyperalgesia induced by repeated acid injections in muscle is
    abolished by the loss of ASIC3, but not ASIC1. Pain 106, 229–239.
    72
    Steen, K.H., Reeh, P.W., Anton, F. and Handwereker, H.O. (1992). Protons selectively induce
    lasting excitation and sensitization to mechanical stimulation of nociceptors in rat skin, in vitro.
    J. Neuronscience 12, 86-93.
    Stucky C. L., and Lewin G. R.( 1999). Isolection B4-poitive and –negative nociceptors are
    functionally distinct. J. Neurosci. 19-15, 6497-6505.
    Sutherland S. P., Benson C. J., Adelmen J. P. and McCleskey E.W.(2001). Acid-sensing ion
    cahnnels 3 matches the acid-gated current in cardiacischemia-sensing neurons. Proc. Natl. Acad.
    Sci. USA, 98 (2), 711-716.
    Tominaga,Makoto., Michael J. Caterina., Annika B. Malmberg.,Tobias A. Rosen., Heather Gilbert.,
    Kate Skinner., Brigitte E. Raumann., Allan I. Basbaum., and David Julius.(1998). The
    Cloned Capsaicin Receptor Integrates Multiple Pain-Producing Stimuli. Neuron, 21, 531–543.
    Ugawa S., Ueda T., Nishigaki M., Shibata Y. and Shimada S.(2002). Amiloride-blockable
    acid-sensing ion channels are leading acid sensors expressed in human nociceptors. J. Clin.
    Invest. 110, 1185-1190.
    Vellani, Vittorio., Olof Zachrisson, and Peter A McNaughton. (2004). Functional bradykinin B1
    receptors are expressed in nociceptive neurones and are upregulated by the neurotrophin GDNF.
    J Physiol. 15; 560, 391–401.
    Walker, Katherine M, WALKER., Laslo Urban, Stephen J.Medhurst., Sadhana Patel, Mohanjit
    Panesar., Alyson J. Fox, and Peter Mcintyre. (2002). The VR1 Antagonist Capsazepine
    73
    Reverses Mechanical Hyperalgesia in Models of Inflammatory and Neuropathic Pain.
    J PET,304, 56-62.
    Wang, J., Kon, J., Mogi, C., Tobo, M., Damirin, A., Sato, K., Komachi, K., Malchinkhuu, E.,
    Murata, N., Kimura, T., Kuwabara, A., Wakamatsu, K., Koizumi, H., Uede, T., Tsujimoto, G.,
    Kurose, H., Sato, T., Harada, A., Misawa, N., Tomura, H. and Okajima, F. (2004). TDAG8 is a
    proton-sensing and psychosine-sensitive G-protein-coupled receptor. J. Biol. Chem. 279,
    5626-45633.
    Weng, Z., Fluckiger, A., Nisitani, S., Wahl, W.I., Le, L.Q., Hunter, C.A., Fernal, A.A., Beau,
    M.M.L. and Witte O.N., 1998. A DNA damage and stress inducible G protein-coupled receptor
    blocks cells in G2/M (1998). Proc. Natl. Acad. Sci. U.S.A. 95, 12334-12339.
    Xu, Y. (2002). Sphingosylphosphorylcholine and lysophosphatidylcholine:G protein-coupled
    receptors and receptor-mediated signal transduction. Biochimica et Biophysica Acta .1582,
    81-88.
    Zaslavsky,Alexander., Lisam, Shanjukumar Singh., Haiyan, Tan., Huawen ,Ding ., Zicai,
    Liang.,Yan Xu. Homo- and hetero-dimerization of LPA/S1P receptors, OGR1 and GPR4 (2006).
    Biochimica et Biophysica Acta. 1761 , 1200–1212.
    Zhu, K., Baudhuin, L.M., Hong, G., Williams, F.S., Cristina, K.L., Kabarowski, J.H.S., Witte, O.N.
    and Xu, Y. (2001). Sphingosylphosphorylcholine and lysophosphatidylcholine are ligands for
    the G protein-coupled receptor GPR4. J.Biol. Chem. 276, 41325-41335.
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    Date of Submission 2007-07-22

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