博碩士論文 100224023 詳細資訊




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姓名 許若姍(Jo-shan Hsu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 ASIC3和TRPV1基因缺乏小鼠在異丙腎上腺素造成心肌缺血後的基因表達
(Gene expression patterns in ASIC3 and TRPV1 deficient mice after isoproterenol-induced myocardial ischemia)
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摘要(中) 胸痛是心肌缺血的標誌,時常發生於心臟時沒有獲取足夠的氧氣時。心肌缺血會引起氫離子 (proton)或緩激肽 (bradykinin)的產生,激活心臟初級傳入纖維,特別由Aδ和C纖維傳入,轉導心臟痛覺至中樞神經系統,從而導致疼痛。在前人研究中指出第三型酸敏感離子通道 (ASIC3)在傳遞心臟感覺的神經束上,可能參與在心肌缺血後的調控,在前人實驗中ASIC3-/-小鼠短暫的心肌缺血時,會使心電圖引起長時間的ST段壓低和心肌纖維化,但不會在ASIC3 +/+小鼠或TRPV1-/-小鼠中發現此作用。然而,分子參與缺血誘導的疼痛是目前還並不明確。
因此在這項研究中,我利用小劑量異丙腎上腺素 (isoproterenol)誘導ASIC3或TRPV1基因剔除的小鼠短暫性心肌缺血,異丙腎上腺素注射後在心臟肌肉會造成短暫的缺氧。在異丙腎上腺素注射後氫離子敏感的G蛋白偶合受體(proton-sensing GPCR)的G2A基因表現增加。在ASIC3基因缺乏小鼠會提早發生心肌缺氧反應,而G2A基因表現量會受到抑制。因此G2A基因可能參與ASIC3調控的途徑,保護心肌細胞以避免受到缺血性造成的損傷。
摘要(英) Chest pain is the hallmark of myocardial ischemia, that often occurs in heart when sufficient oxygen for the needs is not acquired. Myocardial ischemia induces production of proton or bradykinin, which activates cardiac primary afferents, specifically Aδ- and C-fibre afferents, to transduce cardiac nociception to the central nervous system, leading to pain. In previous studies, acid-sensing ion channel 3 (ASIC3) on cardiac sensory afferents to sense protons and some regulations, participate in the regulation of myocardial ischemia. Transient myocardial ischemia induces prolonged ST-segment depression and more severe cardiac fibrosis in ASIC3-/-, but not in ASIC3+/+ or TRPV1-/-. However, molecular involved in ischemia-induced pain is not clear.
In this study, I used low-dose isoproterenol to induce transient myocardial ischemia in ASIC3 or TRPV1 knockout mice. Isoproterenol injection caused transient hypoxia in cardiac muscle. Expression of proton-sensing G-Proton-coupled receptor(GPCR) G2A,was increased after isoproterenol injection. In ASIC3-/- mice, transient hypoxia occurred early and G2A expression was inhibited.
Therefore, G2A may participate in ASIC3-mediated pathway to protect cardiac cells from severe ischemic insults.
關鍵字(中) ★ 心肌缺血
★ 異丙腎上腺素
★ 第三型酸敏感離子通道
★ 辣椒素受體
關鍵字(英) ★ Ischemia
★ Isoproterenol
★ ASIC3
★ TRPV1
論文目次 目錄
中文摘要……………………………………………………………… Ι
英文摘要……………………………………………………………… Π
致謝………………………………………………………………… Ⅲ
目錄…………………………………………………………………… Ⅳ
圖目錄………………………………………………………………… Ⅶ
表目錄………………………………………………………………… Ⅸ

第一章序論 1
1.1 痛覺 (Pain) 2
1.1.2傷害感受性受器 (nociceptor) 3
1.2.心肌缺血引起的缺血性疼痛 3
1.2.1 心肌缺血引起的組織酸化或損傷 4
1.2.2 心肌缺血引發的傷害感受 5
1.3 酸敏感的受體和離子通道 5
1.3.1 辣椒素受體 (TRPV1) 5
1.3.2 第三型酸敏感離子通道 (Acid-sensing ion channel,ASIC3) 6
1.3.3 酸敏感G蛋白偶合受體 (proton-sensing G-protein coupled receptor) 6
1.4 與心肌缺血相關的血清素受體 7
1.5 電壓調控鈣離子通道 (Voltage-gated calcium channel, Cav) 8
1.6研究動機與目的 9
第二章 實驗材料與方法 10
2.1實驗材料 11
2.1.1 實驗小鼠 11
2.1.2實驗藥品 11
2.2實驗方法 12
2.2.1 小鼠基因型的判定 (Genotyping) 12
2.2.2 心肌缺血動物模式 14
2.2.3 組織冷凍切片 16
2.2.4 免疫染色(immunohistochemistry) 16
2.2.5 核醣核甘酸 (RNA)的萃取 17
2.2.6 RNA品質和濃度的測量 19
2.2.6 染色體DNA汙染檢測 (Detection of Genomic DNA contamination) 21
2.2.7 DNase I 的處理 21
2.2.8 cDNA的合成 22
2.2.9 反轉錄-聚合酶鏈鎖反應分析 (RT-PCR) 23
2.2.10 同步定量PCR系統 (Real-Time Quantitative PCR,Q-PCR) 23
2.2.11 統計分析 24
第三章 結果 25
3.1異丙腎上腺素引起心臟肌肉缺氧現象 26
3.2異丙腎上腺素注射後40分鐘會使野生型小鼠心臟的G2A、TDAG8以及ASIC1a基因的表現量增加 26
3.3異丙腎上腺素注射後40分鐘會使野生型小鼠左側背根神經節的 G2A和TRPV1 基因表現量增加與ASIC3基因在早期調控以及在右側注射後40 分鐘TRPV1、ASIC1a和ASIC3表現量增加 27
3.4缺乏酸敏感離子通道3小鼠使用異丙腎上腺素引起心肌缺血在檢測心肌缺氧實驗上並未具有延長缺氧的時間 27
3.5 ASIC3-/- 小鼠心臟G2A、 TDAG8、TRPV1以及ASIC1a基因被抑制增加以及G2A基因早期調控 28
3.6異丙腎上腺素注射後10分鐘會使ASIC3+/+小鼠右側背根神經節 (C8-T3) GRP4表現量增加 28
3.7異丙腎上腺素會造成血清素受體2B基因被抑制增加在ASIC3-/- 小鼠心臟 29
3.8 mCav1.2、mCav1.3、mCav2.3和mCav3.2基因被抑制增加在ASIC3-/-小鼠心臟 29
3.9 異丙腎上腺素造成心臟組織缺氧的情況在TRPV1+/+、TRPV1+/-以及TRPV1-/-小鼠 30
3.10 G2A與ASIC1a基因抑制增加以及GPR4、TDAG8和ASIC3基因增加在TRPV1-/-小鼠注射異丙腎上腺素40 分鐘後的心臟 31
3.11異丙腎上腺素造成TRPV1-/- 小鼠背根神經節左側G2A與GPR4基因表現受到抑制和在40 分鐘後ASIC1a基因增加以及右側G2A、GPR4和OGR1基因表現受到抑制和1天後ASIC1a基因增加 31
3.12異丙腎上腺素注射後40 分鐘會造成血清素受體2A基因被增加在TRPV1-/-小鼠心臟中 32
3.13 異丙腎上腺素會造成TRPV1-/-小鼠在注射後10和40分鐘後使mCav2.2基因表現量增加 32
3.14 ASIC3&TPRV1雙基因剃除小鼠 G2A基因在40 分鐘後被抑制增加 32
3.15 ASIC3與TRPV1雙基因剔除小鼠會造成血清素受體表現量受到抑制 33
3.16 ASIC3與TRPV1雙基因剔除小鼠mCav1.2、mCav2.2 和mCav2.3和基因抑制增加以及在注射40分鐘與1天後mCav3.2基因增加 33
第四章 討論 34
4.1異丙腎上腺素引起心肌缺血的影響 35
4.2 G2A在心肌缺血的影響 36
4.3 缺乏ASIC3基因在心肌缺血G2A與TDAG8基因的影響 37
4.4 TDAG8在心肌缺血過程中的影響 38
4.5 ASIC1a在心肌缺血過程中的影響 38
4.6血清素受體2A、2B和3A 可能會被ASIC3參與異丙腎上腺素引起的心肌缺血所調控 39
4.7鈣離子通道會受到ASIC3基因參與在異丙腎上腺素引起的心肌缺血所調控 40
4.8結論 41
第五章 參考文獻 42
附錄 125
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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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