週邊發炎反應增加酸敏感受體- TDAG8基因在背根神經節之表現量

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陳映儒(Ying-Ju Chen) 查詢紙本館藏

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生命科學系

論文名稱

週邊發炎反應增加酸敏感受體- TDAG8基因在背根神經節之表現量
(Increased expression of a proton-sensing G-protein coupled receptor, TDAG8, in DRG neurons after peripheral inflammation)

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摘要(中)

在週邊發炎反應中，常可發現在受傷組織有因血流速率減低、代謝速率增快、組織滲透壓改變等原因，造成高濃度氫離子累積，而導致局部組織酸化的情形。這些高濃度氫離子會去活化痛覺神經細胞 (small-diameter neurons) 上之正離子通道，如辣椒素受體 (VR1) 或酸敏感受體3 (ASIC3) ，進而造成發炎反應中之痛敏感現象。然而，氫離子除了活化這些相關之離子通道外，同時亦可活化其他G蛋白偶合受體。本篇論文主要即在探討這些受酸活化之G蛋白偶合受體是否亦參與發炎反應中之痛覺相關現象。我們發現，已被證明為受酸性活化之Ｔ-細胞死亡相關受體8 (TDAG8 receptor)有表現在痛覺相關之神經細胞。在週邊發炎反應一天後，Ｔ-細胞死亡相關受體基因8在神經細胞有表現量明顯增加之情形。其中一類痛覺相關之神經細胞其在表面有醣蛋白 (IB4) 結合，已被證明與發炎反應後痛覺敏感之現象有相關性。而這類神經細胞有明顯增加Ｔ-細胞死亡相關受體8之表現量。另外，另一纇在自然情況下非傳遞痛覺之神經細胞 (large-diameter neurons)在發炎反應後亦增加Ｔ-細胞死亡相關受體8之表現量。因此，我們推測Ｔ-細胞死亡相關受體8增加在發炎反應可能與發炎後之痛敏感 (hyperalgesia) 以及觸感痛 (allodynia) 現象有關。另外，高濃度氫離子能刺激大量表現Ｔ-細胞死亡相關受體8之細胞累積環單磷酸腺甘 (cAMP)。因此Ｔ-細胞死亡相關受體8可能在發炎反應中經由環單磷酸腺甘相關路徑，而調控神經細胞內之反應。

摘要(英)

High concentrations of hydrogen ions are usually found in local area surrounding the insulted tissues during several inflammatory responses. The tissue acidosis phenomenon is thought to be associated with inflammatory pain because the proton molecule can activate nociceptors (pain-related neurons) directly through proton-sensing ion channels. Several proton-sensing receptors expressed in dorsal root ganglia neurons. Some of them, such as vallinoid receptor1 and acid-sensing ion channel 3 are thought to be involved in inflammatory hyperalgesia. However, it is still unclear that whether there are any other proton-sensing receptors involved in inflammation. Here we have demonstrated that a proton-sensing G-protein-coupled receptor, mouse T-cell Death Associated gene 8 (mTDAG8), was predominantly expressed in small-diameter neurons, which give rise to the majority of nociceptors. The transcripts of mTDAG8 were increased 24 hours after complete Freund’s Ajuvant (CFA)-induced inflammation, suggesting that the TDAG8 receptors might associate with inflammatory pain. Consistently, data of in situ showed that the total TDAG8-expressing neurons increased approximately 15% in DRG neurons after CFA-inflammation. Most of the increased TDAG8-expressing neurons are large-diameter neurons (9-10%), and the increased small-diameter neurons expressing TDAG8 are restricted in IB4-positive neurons (5-7%). Since the large-diameter neurons can be activated by non-noxious stimulations during inflammation, and the responses of IB4-positive neurons are enhanced after treating with inflammatory mediators, the increased number of neurons expressing TDAD8 receptors may associate with allodynia and hyperalgesia in inflammation. In addition, the mTDAG8-transfected HEK 293 cells accumulated the cAMP in responding to pH 6.0 buffers. Thus the TDAG8 may mediate certain responses through cAMP-pathway in neurons after inflammation.

關鍵字(中)

★ 發炎
★ 背根神經節
★ 組織酸化

關鍵字(英)

★ inflammation
★ dorsal root ganglia
★ tissue acidosis

論文目次

Abstract Ⅰ
摘要 Ⅱ
Acknowledgments Ⅲ
Contents Ⅳ
List of Figures VII
List of Tables IX
Abbreviation XI
Chapter 1 Introduction …………1
1.1 Pain …………2
1.1.1 Nociception and nociceptors 3
1.1.2 Nociceptive pathways 5
1.1.3 Modulations of nociception 6
1.2 Inflammatory pain …………8
1.2.1 Inflammatory mediators 9
1.2.2 Tissue acidosis 11
1.2.3 Other factors involved in inflammatory pain 12
1.3 Proton receptors …………13
1.3.1 Proton-gated ion channels 14
1.3.2 Proton-sensing G-protein coupled receptors 16
1.3.2.1 T cell death-associated gene 8 16
1.3.2.1 Other OGR1 family activated by proton and lysophospholipid 19
1.4 The objective of the thesis ………..22
Chapter 2 Materials and Methods … ..…….23
2.1 Polymerase chain reaction (PCR) …...…….24
2.1.1 Design of primers 24
2.1.2 Preparation of agarose gels and gel electrophoresis 25
2.1.3 General polymerase chain reaction (PCR) 25
2.1.4 Reverse transcription PCR 26
2.1.5 Quantitative PCR (Q-PCR) 26
2.2 Preparation of cDNA templates for RT-PCR and quantitative
PCR experiment …………26
2.2.1 Tissues preparation 27
2.2.2 Extraction of RNA 27
2.2.2.1 Large amounts of RNA 27
2.2.2.2 Small amount of RNA 28
2.2.3 Synthesis of complementary DNA 28
2.3 Amplification and purification of plasmid …………29
2.3.1 Plasmid miniprep midiprep 29
2.4 Cloning and sub-cloning of mouse TDAG8 gene …………31
2.4.1 Preparation of vectors 31
2.4.2 Synthesis of inserts 31
2.4.3 Ligation 31
2.4.4 PCR screening for plasmid containing target gene 32
2.5 In situ hybridization and immunohistochemistry experiments …………32
2.5.1 Preparation of probes for in situ hybridization 32
2.5.2 Prepare the tissues sections 33
2.5.3 Hybridization 34
2.5.4 Immunohistochemistry 35
2.6 Cell culture and transfection …………35
2.6.1 Subculture 35
2.6.2 Transfection 36
2.7 Measurement of accumulated cAMP in cells …………37
2.7.1 Plate coating 37
2.7.2 Cell stimulation 37
2.7.2.1 Treatment of pH buffer 37
2.7.2.2 Pre-treatment of pertussis toxin 38
2.7.3 ELISA set up 38
2.7.3.1 Preparation of ELISA plate and samples 38
2.7.3.2 Set-up of standard curve and antibodies 39
2.7.3.3 ELISA assay 39
2.7.3.4 Calculation of results 40
2.8 Animal experiments …………41
Chapter 3 Results …………42
3.1 Cloning mouse TDAG8 gene 43
3.2 Tissue distribution of mouse TDAG8 gene ASIC3 wild-type
and knockout mice ………….43
3.3. Mouse TDAG8 predominantly expresses in small diameter
DRG neurons ………….45
3.4 The Mouse TDAG8 proteins were predominantly in small-
diameter DRG neurons ………….46
3.5. Mouse TDAG8 gene expressed in both IB4-positive and IB4-negative
neurons 47
3.6. Mouse TDAG8 gene co-expressees with VR1 gene in
small-diameter neurons …………48
3.7 Gene expression changes of the OGR1 family of the CFA,
carrageenan, and capsaicin-induced inflammation. …………49
3.8. The TDAG8 expressing DRG neurons had increase in the
CFA-induced inflammation …………50
3.9. Increase the mTDAG8 proteins in large-diameter DRG
neurons after CFA-induced inflammation. …………51
3.10. Expression of mTDAG8 gene is increased in IB4-
positive neurons among small-diameter neurons …………52
3.11. Increase the expression of TDAG8 gene in both VR1+ and VR1-
small-diameter neurons after CFA-induced inflammation. …………53
3.12. An Increase in intracellular cAMP by proton-stimulation in
mouse TDAG8 -overexpressing HEK 293cells …………52
Chapter 4 Discussion …………56
4.1 Mouse TDAG8 gene expressed in somatic and neuronal tissues …………57
4.2 The expression of TDAG8 is predominantly in IB4-positive
small-diameter neurons …………60
4.3 Increased the expression of mTDAG8 gene 24 hours after CFA
-induced inflammation …………62
4.4 Cyclic AMP accumulated in TDAG8-transfected HEK 293 cells
by stimulating with acidic buffers …………65
4.5 Members of OGR1 family play different roles in peripheral
inflammatory models …………68
References …………70
　　　　　　　　　　　
Appendix ……….122

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指導教授

孫維欣(Wei-Hsin Sun)

審核日期

2006-7-20

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