謝學真臺灣大學：化學工程學研究所賴大林Lai, Da-LinDa-LinLai2007-11-262018-06-282007-11-262018-06-282004http://ntur.lib.ntu.edu.tw//handle/246246/52229Nrf2 is a nuclear factor that can initiate transcription of many antioxidant response element (ARE)-mediated genes including heme oxygenase-1 (HO-1). Nrf2 has been previously found to undergo nuclear translocation in response to shear stress, in which phosphatidylinositol 3-kinase (PI3K) is involved in this process. In this study, it was found that shear stress induced translocation of cytoplasmic Nrf2 into nucleus and increased Nrf2 and HO-1 protein expression in human umbilical vein endothelial cells (HUVECs). The mRNA level of Nrf2 was found to increase in short duration. Moreover, pretreatment of HUVECs with protein synthesis inhibitors cyclohexamide (CHX) abolished Nrf2 protein expression induced by shear stress. However, pretreatment of HUVECs with PKC inhibitor calphostin C and ERK1/2 inhibitor PD98059 had no effect on nuclear translocation of Nrf2 induced by shear stress. We speculate that shear stress increases Nrf2 protein expression in HUVECs by a transcriptional mechanism that also enhances Nrf2-mediated transcriptional activation of many ARE-mediated genes. In contrast, treatment with the proteasome inhibitor (MG132) caused an accumulation of Nrf2 in nucleus and cells. This study also found that reactive oxygen species (ROS) induced the translocation of Nrf2 into nucleus and increased HO-1 protein expression. In addition, treatment with phenolic antioxidant tert-butylhydroquinone (tBHQ) induced remarkably HO-1 protein expression as well as increased the level of Nrf2 in HUVECs. Treatment of HUVECs with CHX resulted in the loss of Nrf2 within 4 h. This effect was reversed by tBHQ, indicating that tBHQ could increase Nrf2 protein stability and prevent it from proteasomal degradation. Taken together, these data suggested that Nrf2 can be regulated and activated by different mechanisms under various stimulations, which include shear stress, ROS, and tBHQ. These regulatory mechanisms include the alteration in nuclear translocation, gene expression and protein stability of Nrf2 and it’s release from Keap1. The activation of Nrf2 may result in the expression of atheroprotective HO-1 protein.中文摘要..................................................I Abstract..................................................III 目錄......................................................V 圖表索引..................................................IX 縮寫及符號說明............................................XII 中英名詞對照..............................................XV 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 2.2 蛋白激酶C (protein kinase C, PKC)................13 2.2.1 蛋白激酶C之生理角色.....................13 2.2.2 剪力對PKC訊息傳導之影響.................16 2.3 活性氧族群 (Reactive Oxygen Species, ROS)........19 2.3.1 活性氧族群之生理意義....................19 2.3.2 活性氧族群之調控........................21 2.4 血紅素氧化酶-1 (Heme Oxygenase-1, HO-1)..........26 2.4.1 血紅素氧化酶-1的功能....................26 2.4.2 血紅素氧化酶-1的調控....................28 2.5 Nuclear factor-erythroid 2 related factor2 (Nrf2).32 2.5.1 Nrf2的功能..............................32 2.5.2 Nrf2訊息傳方面的調控....................33 2.5.3 Nrf2穩定性方面的調控控..................41 2.6 tert-Butyl-hydroquinone (tBHQ)...................45 第三章 實驗材料、儀器、原理及方法 3.1 實驗材料.........................................49 3.1.1 細胞培養及流動實驗所用材料..............49 3.1.2 實驗耗材................................50 3.1.3 西方墨點轉印法所用的材料................51 3.1.4 反轉錄聚合酶連鎖反應法(RT-PCR)所用的材料.52 3.2 實驗儀器.........................................54 3.3 實驗原理與方法..........................56 3.3.1 初級臍帶靜脈內皮細胞培養................56 3.3.2 臍帶靜脈內皮細胞繼代培養................56 3.3.3 流動室之設計............................57 3.3.4 流動實驗................................62 3.3.5 蛋白質含量測定..........................62 3.3.6 細胞內特定蛋白質含量測定: Western blot..64 3.3.7 細胞核內蛋白質的抽取....................64 3.3.8 細胞內Nrf2 mRNA含量之測定：RT-PCR.......65 第四章 實驗結果與討論 4.1 剪力對轉錄因子Nrf2調控之探討.....................69 4.1.1 剪力增加內皮細胞中Nrf2及其下游HO-1的基因表現70 4.1.2 剪力對Nrf2之調控探討....................75 4.1.3 剪力對內皮細胞內之ubiquitination的影響..82 4.2 活性氧族群對Nrf2調控及下游HO-1之探討.............84 4.2.1 活性氧族群活化Nrf2並增加HO-1之表現......84 4.2.2 活性氧族群對Nrf2之調控..................89 4.3 tBHQ對Nrf2調控及下游HO-1表現之探討......91 4.3.1 tBHQ增加Nrf2及下游基因HO-1之表現........91 4.3.2 tBHQ對Nrf2調控之探討....................94 4.4 綜合討論.........................................96 第五章 結論及未來研究方向 5.1 結論.............................................103 5.2 未來研究方向.....................................108 參考文獻...............................................1101601897 bytesapplication/pdfen-US內皮細胞剪力轉錄因子shear stressNrf2endothelial cellsthesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/52229/1/ntu-93-R91524057-1.pdf