博碩士論文 100224015 詳細資訊




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姓名 張崇人(Chung-jen Chang)  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-TDAG8在發炎性疼痛中所扮演的角色
(Roles of T-cell Death-Associated Gene 8 in Inflammatory Pain)
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摘要(中) 長期發炎性疼痛主要是由於皮膚或軟組織受到傷害或是疾病引發的心肌缺血或癌症時所產生。其中內部受損的細胞與免疫細胞會釋放發炎相關介質:如蛋白酶、ATP、氫離子等,去活化或調控這些響應組織的傷害性受器,進而產生訊號並將這些訊號從週邊匯集於背根神經節(DRG)再傳到中樞系統最終傳回大腦,產生疼痛的感覺。然而局部的高濃度氫離子會產生組織酸化的現象,並透過酸敏感離子通道(Proton-sensing ion channel)或G蛋白偶合受體(Proton-sensing GPCR)去調控傷害性感覺神經元,因此被認為是造成疼痛的主要條件。在目前的研究中發現有四個酸敏感G蛋白偶合受體,OGR1、GPR4、G2A和TDAG8,皆會表現在背根神經節上參與疼痛相關的中小型直徑細胞;且亦知道當發炎時背根神經節上的T細胞死亡相關基因8 (TDAG8) 的表現量會增加。由於目前此方面的研究尚不清楚,因此推斷TDAG8與發炎性疼痛是否有直接性的相關聯。在本篇論文中,我利用了RNAi干擾技術來抑制TDAG8的基因表現,並探討TDAG8在疼痛中的功能為何。根據我的結果發現降低TDAG8基因與蛋白質的表現可以完全抑制酸所誘導的機械性痛覺過敏感現象;而由完全弗氏佐劑與海藻醣所引發的長期發炎性痛覺過敏感現象也同樣會受到抑制。透過以上的結果可以表明TDAG8是參與在發炎性疼痛中的。
摘要(英) Chronic inflammatory pain results from inflammation in the skin or soft tissues in response to tissue injury, ischemia or cancer growth. The damaged cells and immune cells release inflammatory mediators such as proteases, ATP and proton to activate or modulate nociceptors, leading to pain. Tissue acidosis (high proton concentration) is the major factor to cause pain by activating proton-sensing ion channels and G-protein-coupled receptors (GPCRs). Four proton-sensing GPCRs, OGR1, GPR4, G2A, and T-cell death-associated gene 8 (TDAG8), are identified and expressed in pain-relevant loci, the dorsal root ganglia (DRG) and TDAG8 expression is increased in inflamed DRG. However, it remains unclear whether TDAG8 is involved in inflammatory pain. In this study, I used the shRNA technique to inhibit TDAG8 gene expression and explore TDAG8 function in pain. I have found that reduction of TDAG8 gene and protein expression completely inhibited acid-induced mechanical hyperalgesia. Hyperalgesia induced by CFA or carrageenan was also reduced. These results suggested that TDAG8 is involved in inflammatory pain.
關鍵字(中) ★ 酸敏感G蛋白偶合受體
★ 發炎性疼痛
★ 長期發炎性疼痛
★ T細胞死亡相關基因8
關鍵字(英) ★ proton-sensing GPCRs
★ inflammatory pain
★ Chronic inflammatory pain
★ TDAG8
論文目次 摘要 III
Abstract IV
第一章 緒論 1
1.1 痛覺 (Pain) 2
1.2痛覺訊息傳遞路徑 (Nociceptive pathway) 3
1.3發炎性疼痛 (Inflammatory pain) 4
1.3.1 組織酸化 (Tissue acidosis) 5
1.3.2 氫離子 (Proton) 5
1.4 G蛋白偶合受體 (G-protein-coupled receptors;GPCR) 6
1.4.1 結構與下游反應路徑 (Structure and downstream signaling pathways) 7
1.4.2 酸敏感G蛋白偶合受體 (Proton sensing G-protein-coupled receptors) 8
1.4.3 TDAG8受體 (T-cell Death-Associated Gene 8) 9
1.5 研究動機與目的 10
第二章 實驗材料與方法 11
2.1 實驗材料 12
2.1.1 菌株 12
2.1.2 細胞株 12
2.1.3 核醣核酸干擾 (RNA interference) 12
2.1.4 實驗動物 12
2.1.5 實驗藥品 13
2.2 實驗方法 13
2.2.1 大腸桿菌的轉型作用 (Transformation) 13
2.2.2 細菌的培養 (Grow bacterial) 14
2.2.3 表現質體的製備 14
2.2.3.1 小量的製備 (Mini-prep.) 14
2.2.3.2 中量的製備 (Midi-prep.) 15
2.2.4 表現質體的確認 15
2.2.4.1 限制酵素分析 15
2.2.4.2 瓊脂醣膠的製備及電泳 16
2.2.5 細胞培養 (Cell culture) 16
2.2.6 轉染作用 (Transfection) 17
2.2.7 流氏細胞儀分析TDAG8沉默效率 (Knockdown efficiency) 17
2.2.8 基因表現分析 18
2.2.8.1 細胞核醣核酸的萃取 18
2.2.8.2 DNase I純化處理 19
2.2.8.3 微量組織核醣核酸的萃取 19
2.2.8.4 染色體去氧核醣核酸 (Genomic DNA) 汙染檢測 20
2.2.8.5 cDNA的合成 20
2.2.9 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 21
2.2.9.1 反轉錄-聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 21
2.2.9.2 同步定量聚合酶連鎖反應系統 (Quantitative PCR, Q-PCR) 21
2.2.10 分析細胞內部cAMP的累積情況 22
2.2.10.1 cAMP 收集 22
2.2.10.2 ELISA (The enzyme-linked immunosorbent assay) 22
2.2.11細胞內鈣離子濃度分析 23
2.2.11.1 玻片的前處理 23
2.2.11.2 鈣離子成像分析法 (Calcium image) 24
2.2.12 組織切片的製作 25
2.2.12.1 玻片的處理置備 25
2.2.12.2 組織包埋及冷凍組織切片及固定 25
2.2.13 免疫染色 (Immunohistochemistry) 25
2.2.14 發炎性疼痛動物模式 26
2.2.15 RNAi 干擾抑制發炎性疼痛 27
2.2.15.1 注射陽離子明膠與TDAG8-shRNA質體的復合物 27
2.2.15.2 抑制發炎性疼痛動物模式 27
2.2.16 痛覺行為測試 (Animals behavior test) 28
2.2.17 小鼠腳掌腫脹測量 29
2.2.18 統計分析 29
第三章 結果 30
3.1 TDAG8受到shTDAG8-cherry-A1、B1、C1的沉默(Knockdown)效率 31
3.2 酸性刺激下細胞內cAMP的累積受到TDAG8基因沉默影響而減少 32
3.3 在酸(pH5.5)刺激下,細胞內鈣離子濃度因 TDAG8沉默現象而減少 34
3.4 TDAG8基因大部分分佈於中小型直徑的DRG神經元 34
3.5 shTDAG8-cherry-B1質體會表現在中小型直徑的DRG神經元 35
3.6 shTDAG8-cherry-B1會降低小鼠體內TDAG8的基因表現 36
3.7 抑制TDAG8表現會降低酸引發的機械性痛覺過敏感現象 37
3.8 抑制TDAG8表現會降低完全弗氏佐劑引發的機械性痛覺過敏感現象 38
3.9 抑制TDAG8表現可以達到長期減緩痛覺過敏感現象 39
3.10 抑制TDAG8的表現無法降低完全弗氏佐劑所引發的腫脹現象 40
3.11 shTDAG8-cherry-A1同樣會降低完全弗氏佐劑引發的疼痛 40
3.12 抑制TDAG8表現會降低海藻醣引發的機械性痛覺過敏感 41
3.13 PKCɛ抑制劑對於抑制TDAG8所減緩的機械性痛覺過敏感有加成效果 41
第四章 討論 43
4.1 TDAG8在酸性的刺激下是以活化Gs蛋白來傳遞下游訊號 45
4.2 B1能有效的抑制小鼠體內的TDAG8基因並完全降低酸所引發的疼痛 46
4.3 TDAG8參與完全弗氏佐劑引發的長期機械性疼痛 48
4.3.1 發炎的前期 (0-3小時) 49
4.3.2 發炎的中期至後期 (>4小時-16天) 50
第五章 參考文獻 52
附錄 61
圖3-1. shTDAG8-A1、B1、C1 質體圖譜 62
圖3-2. TDAG8受到shTDAG8-A1、B1、C1的沉默(Knockdown)效率 64
圖3-3. 酸性刺激下細胞內cAMP的累積受到TDAG8基因沉默影響而減少 67
圖3-4. 在酸(pH5.5)刺激下,細胞內鈣離子釋放因 TDAG8沉默現象而減少 69
圖3-5. TDAG8基因大部分分佈於中小型直徑的DRG神經元 71
圖3-6. shTDAG8-B1質體會表現在中小型直徑的DRG神經元 73
圖3-7. shTDAG8-B1會降低小鼠體內TDAG8的基因表現 75
圖3-8. 抑制TDAG8表現會降低酸引發的機械性痛覺過敏感現象 77
圖3-9. 抑制TDAG8表現會降低完全弗氏佐劑引發的機械性痛覺過敏感現象 80
圖3-10. 抑制TDAG8表現可以減緩完全弗氏佐劑引發的長期慢性疼痛 82
圖3-11. 抑制TDAG8的表現無法降低完全弗氏佐劑所引發的腫脹現象 84
圖3-12. shTDAG8-A1同樣會降低完全弗氏佐劑引發的機械性痛覺過敏感現象 85
圖3-13. 抑制TDAG8表現會降低海藻醣引發的機械性痛覺過敏感現象 87
圖3-14. PKCɛ抑制劑對於抑制TDAG8所減緩的機械性痛覺過敏感有加成效果 89
附錄一、溶液、緩衝液及其他藥劑配方 91
附表一 95
附圖 S-1. 流式細胞儀圈選分析細胞與螢光值校正 98
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指導教授 孫維欣(Wei-hsin Sun) 審核日期 2014-7-8
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