謝學真臺灣大學：化學工程學研究所吳畹儀Wu, Wan-YiWan-YiWu2007-11-262018-06-282007-11-262018-06-282005http://ntur.lib.ntu.edu.tw//handle/246246/52292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.第一章 緒論 1.1 動脈粥狀硬化……………………………… 1 1.2 研究動機與目的…………………………… 5 第二章 文獻回顧 2.1 血管內皮細胞與剪力…………………… 7 2.1.1 內皮細胞之結構…………………………… 7 2.1.2 血管內皮細胞所承受之剪力……………… 8 2.2 MAPK訊息傳導路徑………………………… 16 2.2.1 MAPK signaling pathway………………… 16 2.2.2 剪力對MAPK路徑之調控…………………… 17 2.3 PI3K訊息傳導路徑………………………… 19 2.3.1 PI3K signaling pathway………………… 19 2.3.2 剪力對PI3K訊息傳導之調控……………… 23 2.4 活性氧族群與NADPH oxidase…………… 24 2.4.1 活性氧族群………………………………… 24 2.4.2 NADPH oxidase signaling pathway………30 2.5 Nuclear factor-erythroid 2 related factor 2 (Nrf2)……34 2.5.1 Nrf2之結構與生理功能…………………… 34 2.5.2 Nrf2訊息傳導路徑………………………… 39 第三章 實驗材料、儀器、原理及方法 3.1 實驗材料………………………………………43 3.1.1 細胞培養及流動實驗所用材料…………… 43 3.1.2 實驗耗材…………………………………… 44 3.1.3 西方墨點轉印法所用的材料……………… 45 3.1.4 反轉錄聚合酶連鎖反應法所用之材料…… 46 3.1.5 細胞轉染所用之材料……………………… 47 3.2 實驗儀器…………………………………… 48 3.3 實驗原理與方法…………………………… 50 3.3.1 初級臍帶靜脈內皮細胞培養……………… 50 3.3.2 臍帶靜脈內皮細胞繼代培養……………… 50 3.3.3 流動室之設計……………………………… 51 3.3.4 流動實驗…………………………………… 56 3.3.5 蛋白質含量測定…………………………… 57 3.3.6 細胞內特定蛋白質含量測定: Western blot…… 59 3.3.7 細胞核內蛋白質的抽取…………………… 59 3.3.8 細胞內Nrf2 mRNA含量之測定：RT-PCR……60 3.3.9 以電穿孔方式進行細胞轉染……………… 63 第四章 實驗結果與討論 4.1 不同剪力大小對轉錄因子Nrf2之影響…… 65 4.1.1 不同剪力大小對Nrf2蛋白質表現之影響… 65 4.1.2 不同剪力大小對Nrf2 mRNA表現量之影響 67 4.2 剪力活化Nrf2訊息傳導路徑之探討……… 71 4.2.1 MAPK路徑之探討…………………………… 71 4.2.2 NAC對剪力調控Nrf2訊息傳導路徑之影響 74 4.2.3 eNOS及NO所扮演之角色…………………… 77 4.2.4 PKA路徑之探討………………………………82 4.2.5 NADPH oxidase路徑之探討…………………82 4.3 PMA對Nrf2之調控……………………………86 4.3.1 PMA誘發Nrf2之活化……………………… 86 4.3.2 PMA活化Nrf2訊息傳導路徑之探討……… 88 4.4 綜合討論…………………………………… 94 第五章 結論 5.1 結論………………………………………… 103 5.2 未來研究方向……………………………… 106 參考文獻 …………………………………………………… 1072207472 bytesapplication/pdfen-US剪力內皮細胞Nrf2訊息傳導AREshearendothelial cellssignal transductionthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52292/1/ntu-94-R92524011-1.pdf