博碩士論文 942204008 詳細資訊




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姓名 曾健寧(Jiang-Ning Tzeng)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 酸敏感G蛋白偶合受體在小鼠背根神經節神經元中的訊息傳導路徑
(The signaling pathways of proton-sensing G protein-coupled receptors in primary dorsal root ganglion culture)
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摘要(中) 在組織受傷,發炎反應,或神經受損時,通常會造成局部組織氫離子濃度的上升,稱為組織酸化.此現象通常伴隨著疼痛及疾病的產生.之前的研究也證實氫離子是造成疼痛的ㄧ個重要因子.酸敏感受體3 (ASIC3)及辣椒素受體 (VR1)是氢離子的受體.之前的研究結果指出,當這兩個受體缺失後,並不能完全去除酸所造成的疼痛.因此,有可能還有其他分子參予在其中.最近研究指出,卵巢癌G 蛋白偶合受體1 (OGR1),G蛋白耦合受體4 (GPR4),G2-累積受體 (G2A),及T-細胞死亡相關受體8(TDAG8),是ㄧ個酸敏感G蛋白耦合受體家族.實驗室之前的研究成果指出,卵巢癌G 蛋白偶合受體1家族均表現在神經性的組織,包括背根神經節,也就是感覺神經細胞本體的集結處.在這個基因家族中,以卵巢癌G 蛋白偶合受體1的基因表現量為最高.因此我的實驗目的想知道卵巢癌G 蛋白偶合受體1, G蛋白耦合受體4在背根神經節中的分布情形,以及卵巢癌G 蛋白偶合受體1家族在背根神經節神經細胞中的訊息傳遞路徑.實驗結果發現,卵巢癌G 蛋白偶合受體1和 G蛋白耦合受體4主要表現在不表現神經胜肽(IB4+)的痛覺神經元中,並和酸敏感受體3及辣椒素受體有共同表現的情形.另外,大約31%~40%的神經細胞會表現至少兩個卵巢癌G 蛋白偶合受體1家族成員.而在訊息路徑的實驗中,則沒有切確的結論.
摘要(英) Tissue injury, inflammation, or ischemia usually cause an increase in local proton concentration, which is called tissue acidosis. This phenomenon often companies with disease and painful sensation. The proton has been demonstrated as the main factor of acid-induced pain. Acid-sensing ion channel 3 (ASIC3), a member of ASICs family, and vanilloid receptor 1 (VR1) are the proton-sensing receptors. Deficiency of the two genes can not completely eliminate acid–induced pain. It is possible that other molecules involved in acid–induced pain. OGR1 family that belongs to G protein-coupled receptors including ovarian cancer G protein-coupled receptor 1 (OGR1), G protein-coupled receptor 4 (GPR4), G2A, and T cell death associated gene 8 (TDAG8), has been reported as the proton-sensing receptors. The previous study in our lab has found that OGR1 family members are expressed in neuronal tissues, including dorsal root ganglion (DRG). Among these, OGR1 has the highest gene expression levels. However, whether OGR1 and GPR4 are located in nociceptors and their function in DRG remain unclear. Therefore, the objective of the thesis is to determine localization of OGR1 and GPR4 and to study their signaling pathways in primary DRG culture. I have found that OGR1 and GPR4 were mainly expressed in non-peptidergic, small-diameter nociceptors. Approximately 31%~40% of total DRG neurons contain at least two receptors of OGR1 family. A subset of OGR1 family members were co-localized with ASIC3 or VR1. In primary culture experiments, no clear conclusion was found.
關鍵字(中) ★ 酸
★ 背根神經節
★ 神經元
關鍵字(英) ★ nociception
★ proton
★ DRG
論文目次 Contents
Abstract………………………………………………………………………………………I
Contents…………………………………………………………………………………….IX
Lists of figures…………………………………………………………………………..VIII
List of tables……………………………………………………………………………...XII
Chapter 1 Introduction…………………………………………………………………..1
1.1 Pain……………………………………………………………………………………2
1.2 Nociception…………………...……………………………………………………...2
1.3 Sensory neurons in darsal root ganglia………………………………………….3
1.3.1 Propiocrptors……………………………………………………………………...3
1.3.2 Nociceptors……………………………………………………………………….4
1.4 Nociceptive pathways……………………………………………………………...4
1.5 Tissue acidosis………………………………………………………………………6
1.6 Proton-sensing receptors…………………………………………………………..7
1.6.1 Vanilloid receptor 1 (VR1)………………………………………………………..7
1.6.2 Acid-sensing ion channels (ASICs)………………………………………………7
1.6.3 Proton-sensing G protein-coupled receptors……………………………………...8
1.6.4 The OGR1 family as the receptors of proton……………………………………..9
1.7 The objective of the thesis……………………………………………………….10
Chapter 2 Materials and methods……………………………………………………11
2.1 Tissue preparation……………...…………………………………………………..12
2.2 RNA extraction……………………………………………………………………..12
2.3 Polymerase chain reaction……………………………………………..................13
2.3.1 Design of primers………………………………………………………………..13
2.3.2 Synthesis of complementary DNA……………………………………………...14
2.3.3 Reverse transcription PCR (RT-PCR)…………………………………………...14
2.3.4 Quantitative PCR (Q-PCR)……………………………………………………...15
2.3.5 Preparation of agarose gel and electrophoresis………………………………….15
2.4 Amplification of plasmids………………………………………………………...16
2.5 Cloning of mASIC3 and mG2A………………………………………………....17
2.5.1 Preparation of vectors…………………………………………………………...17
2.5.2 Synthesis of inserts……………………………………………………………....18
2.5.3 Ligation………………………………………………………………………….19
2.5.4 PCR screening…………………………………………………………………...19
2.5.5 Sub-cloning of mASIC3…………………………………………………………20
2.6 In situ hybridization and immunohistochemistry experiments…………......20
2.6.1 Preparation of probes……………………………………………………………20
2.6.2 Slides coating……………………………………………………………………21
2.6.3 Preparation the tissue sections…………………………………………………..21
2.6.4 Hybridization……………………………………………………………………22
2.6.5 Immunohistochemistry………………………………………………………….23
2.7 Primary culture of dorsal root ganglion…………………………………………24
2.7.1 Treatment of cover slips by poly-D-lysine………………………………………24
2.7.2 Isolation of mouse dorsal root ganglion…………………………………………24
2.7.3 Primary culture of DRG neurons………………………………………………..24
2.8 Measurement the change of intracellular calcium concentration…………....25
2.8.1 Preparation of different values of pH buffers……………………………………25
2.8.2 Quantitation of intracellular calcium concentration……………………………..26
Chapter 3 Results………………………………………………………………………...28
3.1 Cloning of mASIC3 and mG2A genes……………………………………….....29
3.2 The change of gene expression levels of mOGR1 and mGPR4 in dorsal root ganglia are not notably between wild type and ASIC3 knockout mice
………………………………………………………………………………………...29
3.3 Mouse OGR1 and GPR4 genes are expressed predominantly in nociceptors………………………………………………………………………...30
3.3.1 Mouse OGR1 and GPR4 are expressed predominantly in small-diameter neurons
………………………………………………………………………………….31
3.3.2 Mouse OGR1 and GPR4 genes are expressed predominantly in IB4-positive neurons…………………………………………………………………………..31
3.3.3 Localization of mouse OGR1 and GPR4 genes with anti-VR1 antibodies in sensory neurons………………………………………………………………….32
3.4 OGR1 family shows high percentage of co-localization………………….....33
3.5 OGR1 family shows high degree of co-localization with ASIC3 in small-diameter neurons…………………………………………………………..34
3.6 The gene expression pattern of epithelial cell differentiation gene (EDG) family in mouse dorsal root ganglia....................................................................35
3.7 Dorsal root ganglia primary neurons can be activated by capsaicin………36
3.8 Dorsal root ganglia primary neurons can be activated by proton stimulation..................................................................................................................37
3.9 Intracellular calcium concentration can be elevated by proton stimulation in primary sensory neurons………………………………………………………38
3.10 Proton-sensing receptors are expressed in primary sensory neurons……39
Chapter 4 Discussion…………………………………………………………………….40
4.1 mOGR1 and mGPR4 are expressed predominatly in non-peptidergic nociceptors………………………………………………………………………….41
4.2 Co-localization of mOGR1 and mGPR4 with VR1………………………….41
4.3 Co-localization of proton-sensing GPCRs with ASIC3……………………..42
4.4 Proton-sensing GPCRs show high degree of co-localization in dorsal root ganglia………………………………………………………………………………...42
4.5 DRG primary neurons are activated by proton………………………………..43
References………………………………………………………………………………...103
Appendix………………………………………………………………………………….111
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指導教授 孫維欣(Wei-Hsin Sun) 審核日期 2007-7-27
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