請用此 Handle URI 來引用此文件:
http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36747
標題: | 內皮細胞中轉錄因子Nrf2受剪力之調控訊息傳導路徑探討 Regulatory Mechanisms of Shear Stress-induced Signal Transduction Pathway of Transcription Factor Nrf2 in Endothelial Cells |
作者: | Wan-Yi Wu 吳畹儀 |
指導教授: | 謝學真 |
關鍵字: | 剪力,內皮細胞,Nrf2,訊息傳導,ARE, shear,endothelial cells,Nrf2,signal transduction,ARE, |
出版年 : | 2005 |
學位: | 碩士 |
摘要: | 轉錄因子Nrf2屬於Cap’n’Collar (CNC) family的一員,在被活化後可以結合至antioxidant response element (ARE)上啟動抗氧化基因包括heme oxygenase-1 (HO-1)、NAD(P)H:quinone oxidoreductase-1 (NQO1)等的表現,以對細胞產生抗氧化的保護功能。
本研究使用人類臍帶靜脈內皮細胞(HUVEC),探討受到剪力作用時內皮細胞中轉錄因子Nrf2的訊息傳導路徑之調控。比較5、12、25 dyn/cm2三種不同大小剪力對細胞的刺激,發現在5 dyn/cm2時細胞中的Nrf2蛋白質表現及移轉入核的量最高,而在12 dyn/cm2時Nrf2 mRNA表現量最高,因此推測在低剪力的狀態下細胞需要較高量的Nrf2表現,提供較佳的抗氧化保護作用,且此一機制可能和Nrf2穩定性的調控密切相關。先前研究曾發現剪力可以經由PI3K活化Nrf2及下游抗氧化基因HO-1的表現,本研究針對相關詳細機制做了更進一步的探討,發現JNK、p38等MAPK相關成員及Akt、eNOS、PKA等PI3K下游蛋白質均不直接參與在剪力活化Nrf2的過程中。當使用長效性NO donor(即NOC18)及短效性NO donor(即SNAP)刺激細胞時,發現Nrf2可以被NO所活化,且此一活化程度與NO釋放在細胞中的濃度有關;然而使用NO清除劑(即PTIO)及eNOS抑制劑(即L-NMMA)均不會對於Nrf2的活化造成抑制,整合相關資料推測剪力所產生的NO量可能相對於NO donor仍太低,不足以藉此造成Nrf2的活化。實驗中也發現,使用reactive oxygen species(ROS)清除劑NAC作用時,可以對剪力誘發的Nrf2蛋白質及mRNA表現量有抑制的現象,推測剪力可能經由增加細胞中ROS含量,提高Nrf2轉錄產生的mRNA表現量進而促成Nrf2的活化。當進一步想要探討NADPH oxidase在剪力活化Nrf2中何種角色時,發現使用的NADPH oxidase抑制劑DPI有其副作用,因此尚未能確認NADPH oxidase在剪力活化Nrf2過程中具有之角色。另外,本研究中亦採用PMA作為刺激物,發現在PMA作用下可以活化Nrf2使其進入細胞核內,且PI3K、PKC及NADPH oxidase均參與在PMA對於Nrf2活化的過程中。 綜合以上實驗結果,推測剪力活化Nrf2的可能機制如下:當細胞遭受氧化逆境刺激時,會先活化細胞膜上的PI3K產生PI(3,4,5)P3再經由一連串的反應活化NADPH oxidase,使ROS含量增加造成細胞中氧化還原狀態改變,再進一步活化Nrf2進入細胞核中與small Maf蛋白質形成heterodimer結合至ARE上,啟動下游抗氧化基因的表現而達到保護心血管的功效。 Nrf2 belongs to the Cap’n’Collar (CNC) family. When activated, Nrf2 can bind to the antioxidant response element (ARE) region and initiate the expression of antioxidant genes such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase-1 (NQO1), and then trigger the antioxidant protection mechanism. In this study, we used the human umbilical vein endothelial cells (HUVECs) to probe into the regulation of shear-induced Nrf2 activation. After exposed to different shear stresses of 5, 12, 25 dyn/cm2, we found that cells exposed to shear stress of 5 dyn/cm2 had the greatest Nrf2 protein expression and nuclear translocation level while the mRNA level seemed to be greatest for cells exposed to shear stress of 12 dyn/cm2, indicating that HUVECs might need higher Nrf2 activation level for better antioxidant protection against low shear stress, also the regulation of Nrf2 stability might play an important role in this aspect. Previously it has been reported that shear stress can induce Nrf2 activation and the downstream antioxidant gene HO-1 expression through PI3K pathway, and in this study we tried to elucidate the detailed mechanism. We discovered that the MAPK family members, JNK and p38, as well as the PI3K downstream targets, Akt, eNOS, and PKA, might not be directly involved in the signaling pathway of shear-induced Nrf2 activation. When applying long-effect NO donor (NOC18) and short-effect NO donor (SNAP) as stimulators, Nrf2 would be activated and the activation level was dependent of the amount of NO released by NO donors. However, the NO scavenger PTIO and eNOS inhibitor L-NMMA exerted no effect on the shear-induced Nrf2 nuclear translocation. With the above information, we speculate that perhaps the amount of NO released by HUVECs exposed to shear stress is still too low in comparison to NO donors, which may not be enough for activation of Nrf2. Moreover, when HUVECs were pretreated with the reactive oxygen species (ROS) scavenger NAC, the shear-induced Nrf2 protein and mRNA level would be both inhibited, which indicates that by increasing the cellular ROS level, shear stress could increase the transcriptional Nrf2 mRNA level, and in turn activate Nrf2. When trying to investigate the effect of NADPH oxidase on shear-induced Nrf2 activation, it was found that the NADPH oxidase inhibitor DPI exhibited some undesirable side-effects, therefore we could not identify the role of NADPH oxidase yet. In addition, PMA was also chosen as an inducer in this study. We demonstrated that PMA could induce Nrf2 nuclear-tranlocation, and PI3K, PKC, and NADPH oxidase might be involved in the PMA-induced Nrf2 activation mechanism. Taken together, we proposed a hypothetical mechanism of shear-induced Nrf2 activation: when HUVECs were exposed to oxidative stress, the membrane-bound PI3K would be first activated, and the PI3K product PI(3,4,5)P3 might in turn activate NADPH oxidase through a series activation of proteins, which could alter the redox-status in cells through the increase of ROS, thus causing Nrf2 to translocate into nucleus and form a heterodimer with small Maf protein. This heterodimer would finally bind to the ARE region, initiating downstream antioxidant genes and hence providing the athero-protective effects. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/36747 |
全文授權: | 有償授權 |
顯示於系所單位: | 化學工程學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
---|---|---|---|
ntu-94-1.pdf 目前未授權公開取用 | 2.16 MB | Adobe PDF |
系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。