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研究生:徐晨峰
研究生(外文):Cheng-Fong Hsu
論文名稱:溫灸在大白鼠內關穴下肌肉和相對應器官心臟刺激自由基產生之影響
論文名稱(外文):Effects of Local Somatothermal Stimulation on Free Radical Formation in the Muscle underlying Acupoint PC 6 and the Corresponding Organ (Heart) in Rats
指導教授:邱仁輝邱仁輝引用關係
指導教授(外文):Jen-Hwey Chiu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:傳統醫藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:143
中文關鍵詞:溫灸活性氧分子一氧化氮熱休克蛋白七十
外文關鍵詞:local somatothermal stimulationreactive oxygen speciesnitric oxideheat shock protein 70
相關次數:
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  • 下載下載:15
  • 收藏至我的研究室書目清單書目收藏:0
在本實驗室過去研究中發現溫灸內關穴(PC 6)會在相對應內臟(心臟)誘導熱休克蛋白七十(Hsp70)表現量增加,並進而保護大鼠心臟避免缺血再灌流傷害。然而在這當中,溫灸作用的機轉卻仍尚未瞭解。因此,本研究的目的是為了測試我們的假設:溫灸刺激內關穴位時,是否在穴位部分產生自由基而引致其相對應內臟器官(心臟)Hsp70的表現及其功能的影響。
研究方法中,在適當麻醉的雄性Sprague-Dawley大鼠上,分別在腹結穴(SP 14)和內關穴(PC 6)施予溫灸一次,平均溫度維持在41~42℃,並各分為五個組別:正常組(0 min)、溫灸後5、15、30和60分鐘,作時間效應探討,並評估穴位下肌肉和相對應內臟(心臟)組織中,自由基產生和其調控Hsp70表現機轉之參數,包括DCFH-DA螢光分析測量以H2O2為主的活性氧分子(ROS)、各種化學分析法測量包括一氧化氮( NO)終產物(NO2─和NO3─)、脂質過氧化物(MDA)、超氧化物歧化酶(SOD)、過氧化氫酶(CAT)和麩胺基硫(GSH)、酵素免疫分析法測量環磷酸鳥苷(cGMP)、西方點墨法測量Hsp70和Nrf2的蛋白表現量;最後則以血清之生化檢測項目CK-MB與Troponin I當作評估心肌損傷的參數。
結果顯示在溫灸腹結穴(SP 14)穴位下肌肉會在溫灸後5分鐘產生ROS、NO終產物(NO2─和NO3─)和MDA,並且會依序在溫灸後5分鐘消耗SOD、溫灸後15分鐘消耗CAT和GSH。此外,在溫灸前給予L-NAME會部分阻斷溫灸誘導Hsp70在穴位下肌肉的表現。而溫灸內關穴(PC 6)在相對應內臟(心臟)可觀察到ROS、cGMP、MDA和Nrf2在溫灸後5分鐘產生,而SOD在溫灸後30分鐘增加。在血清方面,CK-MB和cTnI在溫灸後15分鐘產生。此外,在溫灸內關穴(PC 6)穴位下肌肉也可觀察到ROS、NO和Nrf2在溫灸後5分鐘產生,這也再次證實了溫灸會在穴位下肌肉刺激自由基產生的結果。
本研究的結論是溫灸內關穴(PC 6) 會分別在穴位下肌肉和相對應內臟(心臟)刺激自由基產生,,而且在溫灸前給予L-NAME抑制NO產生會部分阻斷溫灸誘導Hsp70在穴位下肌肉的表現。因此可以間接解釋溫灸調控心臟Hsp70表現達到保護心臟功能的機轉。
Local somatothermal stimulation (LSTS) was demonstrated to increase the expression of myocardial heat shock protein 70 (Hsp70) and protected rat hearts against ischemia-reperfusion injury. However, the exact mechanisms of how LSTS works remains unknown. The aim of this study was to test the hypothesis that LSTS at PC 6 induced free radicals formation in the muscle beneath acupoint and subsequently induced Hsp70 expression in the heart.
LSTS was applied 0.5 cm above and onto acupoint Fujie (SP 14) and Neiguan (PC 6), respectively. Under adequate anesthesia, male Sprague–Dawley rat, were treated with LSTS for 1 dose. Following time intervals of 0 min (no treatment),5-, 15-, 30- and 60- min, respectively, reactive oxygen species (ROS) were measured by 2′,7′-dichlorofluorescin diacetate (DCFH-DA) fluorometric assay. Nitrate and nitrite (nitric oxide (NO) end products), malondialdehyde (MDA) , cyclic guanosine monophosphate (cGMP) and free radical scavengers were measured by commercial available kits. Western blot analysis for Hsp70 and Nuclear factor erythroid 2–related factor 2 (Nrf2) in the muscle and the heart were also analyzed; the parameters such as serum creatine kinase-MB isoenzyme (CK-MB) and cardiac tropnin I (cTnI) were measured to evaluate the myocardial injury.
The results showed that the generation of ROS, NO end products (nitrate and nitrite) and MDA in 5 min-, the consumption of superoxide dismutase (SOD) in 5 min-, and the consumption of catalase (CAT) and glutathione (GSH) in 15 min- after the application of LSTS suggested that there were free radicals formation in the muscle beneath the acupoint area. The LSTS-induced Hsp70 expression in the muscle were partially blocked by L-NAME treatment. Besides, the formation of ROS, MDA, cGMP and Nrf2 in 5 min- , the increase of serum CK-MB and cTnI in 15 min- and subsequently the generation of SOD in 30 min- suggested that there were free radicals formation in the heart after LSTS treatment on left PC 6. Finally, There were also ROS, NO end products (nitrate and nitrite) and Nrf2 expression in 5 min- after LSTS were noticed in the muscle beneath acupoint PC 6.
We concluded that LSTS induced free radicals formation in the muscle beneath the acupoint PC 6 and, the corresponding organ–heart, which might explain the possible mechanisms of LSTS-induced myocardial protection in rats.
目次 Ⅰ
表次及圖次 Ⅸ
中文摘要 ⅩⅢ
英文摘要 ⅩⅤ
第一章 緒論 1
第一節 自由基之概念 2
一、 自由基的產生 3
1. 活性氧分子 (Reactive oxygen species) 3
2. 活性氮分子(Reactive nitrogen species) 7
二、 自由基的氧化傷害 10
三、 自由基與缺血再灌流傷害的關係 12
四、 對抗自由基的防衛機制 13
五、 自由基調控生理反應 15
第二節 熱休克蛋白之介紹及誘發因素 16
一、 熱休克蛋白的功能與調控機制 18
二、 熱休克蛋白家族 20
三、 熱休克蛋白與自由基之關係 23
第三節 中醫觀點 26
一、 灸療的起源與發展 26
二、 針灸穴位相對的特異性 28
1. 中醫古典文獻之描述 28
2. 針灸近代相關之研究 30
三、 溫灸與局部熱效應之關係 31
第四節 本實驗室之溫灸研究成果 32
一、 溫灸誘導一氧化氮釋放對生理功能影響 32
二、 溫灸誘導熱休克蛋白產生對內臟的保護作用 33
第五節 研究動機與目的 35
第二章 實驗材料及方法 36
第一節 實驗動物 37
第二節 動物麻醉 37
第三節 實驗儀器 、藥品試劑與手術器械 37
一、 實驗儀器 37
二、 化學藥品及試劑 39
三、 手術器械 44
第四節 針灸參數的選擇 44
一、 穴位的選擇及解剖定位 44
二、 溫灸的方法與模型之建立 45
1. 溫灸參數科學化 45
2. 溫灸參數 46
3. 劑量效應與時間效應的選擇 47
第五節 實驗設計 47
一、 實驗設計及分組 47
1. 先驅實驗 47
2. 溫灸腹結穴(SP 14)在局部肌肉和心臟產生
自由基之時間效應實驗 48
3. 溫灸腹結穴(SP 14)在局部肌肉一氧化氮影響
熱休克蛋白七十表現量實驗 48
4. 溫灸內關穴(PC 6)在局部肌肉和相對應內臟
(心臟)產生自由基之時間效應實驗 49
第六節 評估方法 50
一、 2′,7′-dichlorodihydrofluorescein diacetate
(DCFH-DA)螢光分析法 50
二、 Nitrate and Nitrite (NO end products) 分析法 52
三、 Malondialdehyde (MDA) 分析法 54
四、 Superoxide Dismutase (SOD) 分析法 55
五、 Catalase (CAT) 分析法 56
六、 Glutathione (GSH) 分析法 57
七、 Cyclic guanosine monophosphate (cGMP) 分析法 59
八、 血清生化檢驗Creatine kinase-MB isoenzyme
(CK-MB)和cardiac Tropnin I (cTnI) 60
九、 Heat shock protein 70 (Hsp70)和Nuclear factor
erythroid 2-related factor 2 (Nrf2)表現量測定 62
1. 肌肉和心臟組織的處理 62
2. 蛋白質定量 62
3. 蛋白質電泳分離 63
4. 西方點墨法 (Western blotting) 64
第七節 實驗數據處理和統計方法 65
第三章 結果 66
第一節 溫灸腹結穴(SP 14)在局部肌肉刺激自由基產生
之時間效應 67
一、 溫灸腹結穴(SP 14)在局部肌肉DCFH-DA
之螢光變化 67
二、 溫灸腹結穴(SP 14)在局部肌肉之NO終產物
(NO2─+NO3─)濃度變化 67
三、 溫灸腹結穴(SP 14)在局部肌肉之MDA濃度變化 68
四、 溫灸腹結穴(SP 14)在局部肌肉之自由基清除者:
SOD、CAT活性與GSH濃度變化 68
第二節 溫灸腹結穴(SP 14)在局部肌肉誘發NO影響Hsp70
表現量變化 70
第三節 溫灸內關穴(PC 6)在相對應內臟(心臟)刺激自由基
產生之時間效應 70
一、 溫灸內關穴(PC 6)在相對應內臟(心臟)DCFH-DA
之螢光變化 70
二、 溫灸內關穴(PC 6)在相對應內臟(心臟)之NO
終產物(NO2─+NO3─)濃度變化 71
三、 溫灸內關穴(PC 6)在相對應內臟(心臟)之cGMP
濃度變化 71
四、 溫灸內關穴(PC 6)在相對應內臟(心臟)之MDA
濃度變化 72
五、 溫灸內關穴(PC 6)在相對應內臟(心臟)之自由基
清除者:SOD、CAT活性與GSH濃度變化 72
六、 溫灸內關穴(PC 6)對心肌的損傷指數之影響:
CK-MB、cTnI之濃度變化 74
七、 溫灸內關穴(PC 6)在相對應內臟(心臟)之Nrf2
表現量變化 75
第四節 溫灸內關穴(PC 6)在局部肌肉刺激自由基產生
之時間效應 75
一、 溫灸內關穴(PC 6)在局部肌肉DCFH-DA
之螢光變化 75
二、 溫灸內關穴(PC 6)在局部肌肉之NO終產物
(NO2─+NO3─)濃度變化 76
三、 溫灸內關穴(PC 6)在局部肌肉之Nrf2表現量變化 76
第五節 溫灸腹結穴(SP 14)在心臟刺激自由基產生之
時間效應 76
一、溫灸腹結穴(SP 14)在心臟DCFH-DA之螢光變化 77
二、溫灸腹結穴(SP 14)在心臟之cGMP濃度變化 77
第四章 討論 78
第一節 實驗方法之討論 79
一、 麻醉藥物的使用 79
二、 實驗模型的探討 79
1. 溫灸參數的採用與實施 79
2. 穴位的選擇與其相關性 80
3. 正向控制組結果的探討 81
三、活性氧分子(ROS)分析方法的探討 81
第二節 溫灸刺激自由基產生之時間效應探討 81
一、 時間點的設定 81
二、溫灸腹結穴(SP 14)在局部肌肉刺激自由基產生
之時間效應探討 82
三、溫灸內關穴(PC 6)在局部肌肉和相對應內臟
(心臟)刺激自由基產生之時間效應探討 84
四、溫灸腹結穴(SP 14)在心臟刺激自由基產生之
時間效應探討 86
第三節 與其他熱療比較的異同點 87
一、 相異點 87
1. 方法 87
2. 前置處理和觀察時間 87
3. 機轉層面 88
二、 相同點 88
1. 機轉層面 88
2. 功能層面 89
第四節 體表內臟反射(somato-visceral reflex) 90
第五節 溫灸內關穴(PC 6)調節相對應內臟(心臟)功能
機轉探討 91
第六節 臨床應用與發展 93
第七節 未來與展望 94
第五章 結論 95
第六章 參考文獻 97
圖表附錄 115
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