博碩士論文 92224020 詳細資訊




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姓名 黃佳瑋(Chia-Wei Huang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酸敏感的G蛋白偶合受體─OGR1表現在背根神經節內與痛覺相關的感覺神經元上
(The expression of proton-sensing G-protein-coupled receptor, OGR1, in pain-related neurons.)
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摘要(中) 當組織受傷以及發炎時,局部的氫離子濃度會上升(稱為組織酸化),並伴隨疼痛的感覺。組織酸化是造成疼痛的主要因素。VR1和ASICs家族中的ASIC3已被證實和酸引起的疼痛相關。但是剔除VR1基因或是ASIC3基因並不能抑制酸引起的疼痛。因此,酸敏感G蛋白偶合受體是否參與酸引起的痛覺是非常有趣的研究方向。酸敏感偶合受體包括OGR1、GPR4、G2A和TDAG8,他們原本被證明是溶血磷脂質(lysophospholipids)的受體。我使用反轉錄聚合酶連鎖反應(RT-PCR)以及定量聚合酶連鎖反應的檢測方法發現在小鼠中,OGR1、GPR4、G2A和TDAG8這四個基因都表現在背根神經節(dorsal root ganglion)。在這四個基因中,OGR1的基因表現量是最高的,這表示OGR1可能在感覺的反應中具有功能。我使用同位雜交法檢驗GR1基因分布在背根神經節的哪些神經元中。結果發現,在背根神經節中的小細胞有百分之三十五表現OGR1基因,而背根神經節中的大細胞有百分之二十一表現OGR1基因。因為小細胞和痛覺相關,所以OGR1的主要功能可能和痛覺相關。氫離子和sphingosylphosphatidylcholine (SPC,一種溶血磷脂質)都可以活化OGR1,使細胞內的鈣離子濃度增加,且氫離子和SPC是相互競爭的agonists。
摘要(英) Tissue injury and inflammation often raise local proton concentration (called tissue acidosis) and accompany with painful sensations. Tissue acidosis is a dominant factor that contributes to pain. Vanilloid receptor 1 (VR1) and acid-sensing ion channel 3 (ASIC3), one member of ASIC family, are proved to be related to acid-induced pain. However, acid-induced pain is not inhibited in ASIC3 or VR1 gene deletion. Therefore it would be interesting to know whether proton-sensing GPCRs are involved in acid-induced nociception. Proton-sensing GPCRs, including ovarian cancer G-protein-coupled receptor 1 (OGR1), GPR4, G2A, and T cell death associated gene 8 (TDAG8), are originally identified as lysophospholipid receptors. Using RT-PCR and quantitative PCR, I have found that mouse OGR1, GPR4, G2A, and TDAG8 are expressed in dordal root ganglion (DRG). Among the four genes, OGR1 has the highest expression levels in DRG, suggesting that OGR1 may have a role in sensory responses. The localization of OGR1 gene in DRG neurons was examined using in situ hybridization and the results show that 35% small-diameter and 21% large-diameter neurons have OGR1 expression. Since small-diameter neurons are related to nociception, the major function of mOGR1 is probably involved in nociception. Both of proton and sphingosylphosphatidylcholine (SPC) can activate OGR1 to increase intracellular calcium concentration, and they are competitive agonists for OGR1.
關鍵字(中) ★ G蛋白偶合受體
★ 背根神經節
★ 痛覺
★ 神經科學
關鍵字(英) ★ dorsal root ganglion
★ nociception
★ pain
★ neuroscience
★ G-protein-coupled receptors
★ OGR1
論文目次 Contents
Contents……………………………………………….........................I
Lists of figures …………….………………………………...…...…..V
Lists of tables………………………………………………………..VІ
Abbreviation………………………………………………………..VII
Chapter 1 Introduction…………………….……….…………...…...1
1.1 Pain and nociception……………………………………...…...2
1.1.1 Nociceptors……………………………..…………….........2
1.1.2 Locations of nociceptor terminals in spinal cord……..................4
1.1.3 Ascending pathways from spinal cord to brain……….............…5
1.2 Tissue acidosis and inflammatory pain …………….………….…..6
1.2.1 Tissue acidosis and pain…………...……..……………...….6
1.2.2 Inflammatory pain and tissue acidosis..……………...…….....7
1.3 Proton-sensing receptors…..……………………………...…...9
1.3.1 Acid-sensing ion channels (ASICs)……………….…….........9
1.3.2 Vanilloid receptor 1 (VR1)………………………................10
1.4 The ovarian cancer G-protein-coupled receptor 1 (OGR1) family.………………………………………………….……11
1.4.1 The physiological roles of sphingosylphophatidylcholine (SPC) and lysophosphatidylcholine (LPC)…………………………...11
1.4.2 The OGR1 family as lysophosphatidylcholine (LPC) receptors…………………………………..…………..13
1.4.3 The OGR1 family as proton receptors…………..……..…….15
1.5 The objective of the thesis………………………..………......16
Chapter 2 Materials and Methods……………………….………....17
2.1 Preparation of solutions………………………………………18
2.2 Preparation of agarose gels and gel electrophoresis………….18
2.3 Polymerase chain reaction (PCR)…………………………….19
2.3.1 General PCR……….………………………………….….19
2.3.2 Reverse transcription PCR (RT-PCR)……………..................19
2.3.3 Quantitative PCR (Q-PCR)………………...………............20
2.3.4 Design of primers…………………………...……….……20
2.3.5 Synthesis of first strand cDNA………………..……………22
2.4 Amplification and purification of plasmids…………………..22
2.4.1 Transformation…………………………………..….........22
2.4.2 Overnight culture and freezing stocks of bacteria...…………..23
2.4.3 Plasmid miniprep and midiprep……………………….........23
2.5 Cloning of mouse OGR1 and GPR4 genes……………….......24
2.5.1 Preparation of vectors………………………………...……..24
2.5.1.1 By specific restriction enzymes…………………..…..24
2.5.1.2 Preparation of T vectors...............................................25
2.5.2 Synthesis of inserts..............................................................25
2.5.3 Ligation.............................................................................26
2.5.4 PCR screening....................................................................26
2.5.5 Subcloning of mOGR1 and mGPR4 genes……..……..……..27
2.6 Tissues preparation...................................................................27
2.7 Extraction of RNA……………………………………………28
2.7.1 RNA extraction using TRIzol reagent……..…………...……..28
2.7.2 RNeasy kit………………………………...……………..28
2.7.3 Measurement of RNA quality and concentration……………...29
2.7.4 Treatment of genomic DNA contamination…………...……...30
2.7.5 Purification of nucleic acid using phenol/chloroform method…..30
2.8 In situ hybridization…………………………………………..31
2.8.1 Preparation of probes for in situ hybridization……………....…31
2.8.2 Slides coating………………………………………..….....32
2.8.3 Cryosectioning…………………………………..……...….33
2.8.4 Fixation and acetylation of the DRG tissue sections…………....33
2.8.5 Hybridization…………………………………....................33
2.9 Cell culture and transfection………………………………….36
2.9.1 Subculture……………………………………………..…36
2.9.2 Treatment of coverslips with poly-D-lysine…………..………37
2.9.3 Transfection……………………………………...…....….37
2.10 Quantitation of intracellular calcium concentration...............38
Chapter 3 Results…………………………..………………………..41
3.1 Four proton-sensing G-protein coupled receptors……………42
3.2 Cloning of mOGR1 and mGPR4 genes………………………43
3.3 Tissue distribution of the mOGR1 family…………………….44
3.4 Mouse OGR1 is the dominant proton-sensing G-protein- coupled-receptor in neuronal tissues……………………….45
3.5 Mouse OGR1 is expressed in both large- and small-diameter neurons in DRG……………………………………………..47
3.6 Proton is a ligand of OGR1…………………………………...48
3.7 Sphingosylphosphatidylcholine (SPC) is a ligand of OGR1....50
3.8 Effects of sphingosylphosphatidylcholine on proton-inducing signaling……………………………………………………..51
3.9 No significant change in proton-sensing GPCRs expression in carrageenan-induced inflammation…………………………….52
Chapter 4 Discussion……………………..……………....................53
4.1 Physiological roles of mOGR1……………………………….54
4.2 The effects of proton and SPC in mOGR1…………………...56
4.3 The inflammation experiments……………………………….59
References……………………………………………………………81
Appendix………………………………….………………………….88
Lists of figures
Figure 3-1 Alignment of amino acid sequences and the phylogenic tree for the mOGR1 family……………………………………61
Figure 3-2 Maps of mOGR1 and mGPR4 constructs.………………..63
Figure 3-3 Gene expression pattern of proton-sensing GPCRs………64
Figure 3-4 Gene expression levels of the mOGR1 family in wild type neuronal tissues……………………………….………...65
Figure 3-5 Comparison of gene expression levels of the mOGR1 family in wild type and ASIC3 knockout mice……………..………66
Figure 3-6 Mouse OGR1 is expressed in both N52-positive and peripherin-positive cells.……………….....…………....68
Figure 3-7 The pH-dependent responses in mOGR1-transfected HEK293T cells..............................................................69
Figure 3-8 Sphingosylphosphatidylcholine dosage responses in mOGR1-overexpressing HEK293T cells…...……..…70
Figure 3-9 Endogenous proton-sensing receptors and epithelial differentiation gene receptors in HEK293T cells….....71
Figure 3-10 Effect of sphingoshylphosphatidylcholine (SPC) in mOGR1-mediated proton signaling………..……….72
Figure 3-11 Effect of SPC in mOGR1-mediated proton signaling....…73
Figure 3-12 No significant change in gene expression of the mOGR1 family after injection of carrageenan.………….………..74
List of tables
Table 2-1 Oligonucleotide primers used in PCR experiments...……...75
Table 3-1 Gene expression levels of the mOGR1 family in different neuronal tissues………………………………………….....76
Table 3-2 Gene expression levels of the mOGR1 family after carrageenan injection…………………..……………..79
Table 3-3 The cell populations expressing mOGR1 in wild type DRG…………………………………………………….80
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指導教授 孫維欣(Wei-Hsin Sun) 審核日期 2006-1-13
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