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研究生:林亞璇
研究生(外文):Ya-Shuan Lin
論文名稱:甲基安非他命誘發H9c2心肌細胞表現第一型血基質氧化酶之機制探討
論文名稱(外文):The mechanism of methamphetamine-induced heme oxygenase-1 expression in H9c2 cardiomyocytes
指導教授:嚴錦城顏鴻章顏鴻章引用關係
指導教授(外文):Jiin-Cherng YenHung-Tsang Yen
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
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:2014
畢業學年度:102
語文別:中文
論文頁數:67
中文關鍵詞:心肌細胞活性氧化物甲基安非他命第一型血基質氧化酶Akt
外文關鍵詞:cardiomyocytesreactive oxygen speciesmethamphetamineHO-1Akt
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甲基安非他命是一種精神興奮劑,但在世界各地廣泛被濫用,會使神經系統受到影響,但其對人體的危害不只限於中樞神經系統,在過去的文獻指出,使用過量的甲基安非他命會造成心律不整導致心臟衰竭或死亡。本研究期望能找出相對應之細胞保護機制,在未來針對甲基安非他命而造成心肌損傷之病患可利用此機制採取治療方式。
  在本研究中發現,甲基安非他命會使H9c2心肌細胞活性氧化物 (Reactive Oxygen Species, ROS) 增加,進而造成細胞凋亡。本研究進一步發現,處理甲基安非他命24小時後,第一型血基質氧化酶 (Heme oxygenase-1, HO-1) 蛋白質表現量顯著上升,預處理HO-1抑制劑tin protoporphyrin (SnPP) 會使甲基安非他命所誘發之H9c2心肌細胞凋亡顯著增加,說明HO-1扮演抗細胞凋亡之角色。為了進一步釐清HO-1被誘發的來源是否由ROS而來,因此分別預處理抗氧化劑Vitamin C、N-acetyl cysteine (NAC) 之實驗組別中也發現,甲基安非他命會使ROS增加,在預處理NAC及Vitamin C後能阻擋甲基安非他命所誘發之HO-1蛋白表現量。本研究也發現,NADPH oxidase抑制劑apocynin無法抑制甲基安非他命所誘發之HO-1,暗示NADPH oxidase可能不參與在甲基安非他命所誘發之ROS產生。
  而根據文獻指出,PI3K/Akt路徑也具有抗細胞凋亡之作用,且在實驗室過去的研究中發現,甲基安非他命在SH-SY5Y神經細胞中可透過PI3K/Akt路徑活化 HO-1的蛋白質表現。研究結果顯示,處理甲基安非他命於ve心肌細胞後,確實可在1小時內明顯活化Akt,接著利用PI3K抑制劑wortmannin的情況下,釐清PI3K/Akt路徑具有細胞保護作用。而在預處理Akt抑制劑API-2的實驗組別中也更加驗證Akt能對抗甲基安非他命所誘發之H9c2心肌細胞凋亡。本研究也進一步探討Akt是否能透過ROS所活化,因此在預處理抗氧化劑Vitamin C及NAC後能阻擋甲基安非他命所誘發之Akt活化,可得知甲基安非他命誘發Akt之活化會透過ROS而來。最後,利用預處理PI3K抑制劑wortmannin及轉染Akt的方式觀察Akt對HO-1的調控情形,研究結果顯示,PI3K/Akt的活化能調控HO-1之蛋白質表現量並達到抗細胞凋亡的效果。
  總結以上的結果發現,處理甲基安非他命除了會使H9c2心肌細胞之ROS增加造成細胞凋亡外,也會啟動另一條保護路徑來對抗本身造成的傷害,而甲基安非他命會透過ROS的活化啟動PI3K/Akt路徑以誘發HO-1蛋白質表現,並達到抗細胞凋亡之保護作用。

Acute adverse effects of methamphetamine (METH) on human health have been mainly attributed to the insults of nervous system. To date, very limited information regarding detrimental effects of acute METH exposure on the cardiovascular system is available. Nevertheless, the molecular and cellular mechanisms by which METH induces cardiotoxicity remain unclear. This study was thus aimed to uncover the endogenous mechanisms and find potentially effective protection approach against the METH-induced cell damages in H9c2 cardiomyocytes.
  In this study, METH caused a rapid increase of reactive oxygen species (ROS) and resulted in apoptosis in H9c2 cardiomyocytes. The level of HO-1 expression was dose-dependently elevated by METH. Moreover, METH-induced decline in cell viability and increase in cleaved caspase-3 level were enhanced by pretreatment with the HO-1 inhibitor tin-protoporphyrin (SnPP). These results suggest a protective role for HO-1 in the METH-induced apoptotic death of H9c2 cardiomyocytes. Following pretreatment with antioxidants of Vitamin C and N-acetyl cysteine (NAC) to decrease the level of HO-1, significantly reduce in HO-1 levels were observed in METH treated H9c2 cells. However, METH-induced decrease in the cell survival rate could not be attenuated by apocynin, an inhibitor of NADPH oxidase (NOX) which plays a major role in ROS production in cardiomyocytes.
  PI3K/Akt pathway was demonstrated to play protective roles in cardoimyocytes. Previous studies in our lab found that METH induced HO-1 expression via activation of PI3K/Akt pathway in SH-SY5Y neuronal cells. The results showed that Akt was significant trend for activated after 1-hour exposure to METH. As was expected , METH-induced apoptosis of H9c2 cardiomyocytes was inhibited by wortmannin, a PI3K inhibitor, suggesting the involvement of PI3K/Akt signaling pathway in METH-induced apoptosis. The Akt inhibitor, API-2, further confirmed the anti-apoptotic role of Akt after METH exposure. Moreover, the increase of ROS under METH treatment may activate Akt which was proven by the addition to Vitamin C and NAC pretreatment followed by METH stimulation. Last, HO-1 protein levels were also regulated by pretreatment of wortmannin. Overexpression of Akt significantly increased the HO-1 level accompanied by a decrease in the activation of caspase-3 in H9c2 cardiomyocytes.
  In conclusion, these results suggest that METH-induced ROS resulted in apoptosis could be attenuated by induction of HO-1 via PI3K/Akt signaling in H9c2 cardiomyocytes. 
致謝........ i
目錄...... ii
中文摘要.............................................. …iii
Abstract ..... v
研究背景................................................ 1
一、甲基安非他命對心肌細胞的毒性作用 .................... 1
二、 活性氧化物 對心肌細 胞的影響 ........................ .. 2
三、細胞自我保護機制 3
四、 HO -1對心肌細胞的保護作用 對心肌細胞的保護作用 ... 4
五、 PI3K/AktPI3K/AktPI3K/Akt 對心肌細胞的調控功能及抗凋亡機制 對心肌細胞的調控功能及抗凋亡機制 .............. ..... 5
研究目的.......................................... 7
材料與方法.......................................... 8
研究結果...................................... 17
討論................................ 23
結論........... 33
參考文獻........................... 28
附圖........ 37
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