血管病變的發生過程被認為起因於血管內皮細胞受到氧化逆境影響的結果，例如動脈粥狀硬化和血管再狹窄。而使血管內皮細胞處於氧化逆境中，是由於細胞內不平衡的氧化還原狀態所造成的，因此這被認為與這些血管病變的發展有相關聯。活化轉錄因子3 (ATF3)是氧化逆境反應基因群之一，ATF3及其目標基因-基質金屬蛋白酵素-2(MMP-2)的活性，皆在動脈粥狀硬化和血管再狹窄的發展中扮演一重要角色。然而，在血管內皮細胞中氧化還原狀態的不平衡，乃造因於過度生成活性氧/氮族群(ROS/RNS)或是抗氧化逆境的系統機能不足，而引發ATF3與MMP-2的調控機制失衡，然而是否為造成血管病變的成因之一，仍然有待釐清。本篇論文藉由使用一氧化氮(NO)、15-deoxy-D12,14-PGJ2 (15d-PGJ2)，以及介白素-1b (IL-1b)來產生氧化逆境衝擊，以探討在此氧化逆境衝擊影響下血管內皮細胞中的ATF3 和MMP-2之調控機制。研究結果顯示，所產生的氧化逆境衝擊誘發了內皮細胞ATF3表現的增加，進而抑制MMP-2的表現。
由於MMP-2持續性生成於內皮細胞中，因此研究NO 對於內皮細胞中MMP-2表現的影響具有其意義。以NO處理內皮細胞，隨著其處理濃度的增加而增加了其抑制MMP-2 mRNA表現的效果。另外以腺病毒帶有內皮細胞一氧化氮合成酶 (eNOS) 基因感染內皮細胞，亦可以減少MMP-2 mRNA的表現。又因為NO可以減低MMP-2啟動子的活性，顯示NO抑制MMP-2表現是藉由抑制其轉錄機制所致。再者藉由zymographic 測定，亦顯示其結果的一致性，即是NO 減少內皮細胞MMP-2的分泌。為了進一步了解NO抑制MMP-2轉錄機制，故分析可能具有活化MMP-2啟動子的轉錄因子結合位置，其結果顯示在全長1716 bps的MMP-2啟動子中介於-1659 與-1629間有p53的結合位置，對於MMP-2啟動子的活性具有決定性的調控作用。若外加NO供給(NOC18)或腺病毒帶有eNOS基因感染的方式，用以增加內皮細胞中的NO，皆可以增加ATF3的表現。另一方面，以帶有ATF3基因的質體轉染或是以MG-132(ATF3 activator)刺激的方式處理內皮細胞，用以增加內皮細胞中ATF3的表現，其結果顯示能夠壓抑MMP-2啟動子的活性，因此這研究結果與文獻報告所指，ATF3具有壓制p53轉錄活性的功能，具有一致性。基於以上的研究結果，無論以外加NO供給者(NOC18)、使用MMP-2抗體、或是以MG-132刺激的方式處理內皮細胞，皆具抑制內皮細胞遷移的類似現象。這些結果顯示，對於在NO 抑制內皮細胞遷移現象的分子機制中，NO可能是藉由增加ATF3來壓抑MMP-2的表現，其扮演一定程度的重要性。
15d-PGJ2是花生四烯酸(arachidonic acid; AA)的衍生物，具有活化過氧化小體增生接受子-g (peroxisome proliferator-activated receptor-g; PPAR-g)的功能。PPAR-g是nuclear hormone receptor 家族的一員，其轉錄的活性則取決於是否與其ligand (例如15d-PGJ2) 結合。研究顯示在動脈粥狀硬化區域中，有大量PPAR-g表現在血管細胞中，例如內皮細胞、單核細胞/巨噬細胞以及平滑肌細胞。另有研究顯示，15d-PGJ2 會增加NO的釋放、促進ATF3的表現以及抑制細胞的遷移。因此本研究首先亦以15d-PGJ2處理的內皮細胞，結果顯示其抑制MMP-2 mRNA的程度是隨著時間增加而增加。又因為15d-PGJ2可以減低MMP-2啟動子的活性，且使用actinomycin D亦無法影響其抑制MMP-2的mRNA表現，所以15d-PGJ2是藉由抑制MMP-2啟動子活性的機制所致。再者zymographic 分析，亦顯示其結果的一致性，即15d-PGJ2 減少內皮細胞MMP-2的分泌。接著15d-PGJ2的處理，增加了內皮細胞中eNOS 的活性以及增加NO的釋放，而若事先以L-NAME (eNOS inhibitor)處理內皮細胞，可以抑制15d-PGJ2所誘發NO之釋放增加的結果。15d-PGJ2亦增加ATF3的表現，分別隨著其處理的濃度以及時間的增加而增加。進一步研究15d-PGJ2對訊號傳遞路徑之影響，顯示無論是以外加p38抑制劑 (SB203580)或是以dominant negative mutant of p38質體轉染方式做前處理，皆可以抑制15d-PGJ2所誘發ATF3的表現。因此這些結果顯示，15d-PGJ2誘發了ATF3的表現，進而抑制MMP-2的生成，其中活化p38訊號傳遞路徑以及增加NO的釋放量，皆有參與其調控的機制。
依據有關介白素-1b(IL-1b)在動脈粥狀硬化致病過程的研究中，顯示IL-1b亦扮演一調控者的角色。在內皮細胞以及平滑肌細胞的研究皆顯示，IL-1b 的處理可以增加細胞內ROS的生成。目前仍未確知內皮細胞經IL-1b 處理後，是否可以產生如上述之類似結果？此外，經由ROS誘發ATF3而抑制MMP-2的表現以及其分子調控機制為何？仍然有待釐清。所以首先證明使用IL-1b處理的內皮細胞，其ATF3的表現，分別地隨著處理的濃度的增加以及時間的增長而增加。另一方面，經由IL-1b處理的內皮細胞中ROS有顯著的增加。若以抗氧化劑(N-acetyl-L-cysteine; NAC)做前處理，則可抑制IL-1b所誘發之ATF3表現。這結果顯示ROS參與了IL-1b所誘發的ATF3表現。另外，藉由Rac1 pull-down 分析，結果顯示IL-1b亦誘發Rac1的活化且需要ROS的參與。若暫時性轉染含有dominant negative mutant of Rac1(RacN17)的質體至內皮細胞中，則可壓制IL-1b所誘發ATF3表現的效果，且若是轉染dominant positive mutant of Rac1(RacV12)質體至內皮細胞中，則可增加其ATF3的表現。所以ROS增加Rac1的活化而參與了IL-1b所誘發的ATF3表現。接著以暫時轉染含有MEKK-1(是一JNK的kinase)的質體於內皮細胞中，使過量表現MEKK-1亦可以增加ATF3的表現。而若以兩種主要JNK磷酸化的抑制劑(SP600125和curcumin)做前處理內皮細胞，結果顯示均可以抑制IL-1b所誘發ATF3表現的效果，但是若以SB203580(p38磷酸化的抑制劑)或是PD98059(ERK磷酸化的抑制劑)做前處理，則無法有效抑制IL-1b所誘發ATF3表現的作用，因此，推斷JNK訊號傳遞路徑的被活化扮演著主要傳遞IL-1b調控ATF3的表現之訊號。再則IL-1b所誘發之ATF3表現減少了內皮細胞MMP-2的分泌，這與前述之結果有其一致性。這部分的結果顯示了，IL-1b處理的內皮細胞，藉由增加ROS的生成 和Rac1的活性，再經由活化JNK訊號傳遞路徑而誘發ATF3的表現，最後導致MMP-2的表現受抑制。
綜觀整個研究的結果可獲以下結論，氧化逆境中的內皮細胞雖然可經由不同的訊號傳遞路徑來增加ATF3的表現，然而所增加的ATF3皆可以抑制MMP-2的表現，進而影響了內皮細胞的遷移。本論文推斷內皮細胞反應氧化逆境時，具有採行一共通性分子調控機制的特性，本研究亦彰顯在血管病變的過程中，維持自由基平衡的重要性。

The development of vascular diseases, such as atherosclerosis and restenosis, is a result of implication to vascular wall initiated by impaired endothelial function in which oxidative stress, an imbalance of redox status, plays a role. Activating transcription factor 3 (AFT3), an oxidative-stress response gene and the target gene matrix metalloproteinase-2 (MMP-2) have been correlated to the development of atherosclerosis and restenosis. Imbalance of redox status, due to either an over-production of reactive oxygene species/reactive nitrogen species (ROS/RNS) or deficiency in antioxidant mechanism, contributes to the expression of ATF3 and MMP-2 in ECs. However, detailed mechanism remains elusive. In the present study, HUVECs (human umbilical cord vein endothelial cells) under oxidative stress induced by nitric oxide (NO), 15-deoxy-△12，14-prostaglandin J2 (15d-PGJ2), and interleukin-1β(IL-1β) were studied for ATF3 and MMP-2 expression. Results showed that ECs under oxidative stress increase ATF3 expression that consequently suppresses MMP-2 gene expression.
MMP-2 is constitutively expressed by ECs. The effect of NO on MMP-2 expression was examined. A dose-dependent inhibition of MMP-2 mRNA level was demonstrated in ECs treated with NO. ECs infected with adenovirus carrying eNOS (Ad-eNOS) reduced MMP-2 expression. The inhibitory effect of NO on MMP-2 expression was a transcriptional event since NO reduced MMP-2 promoter activity. Treatment of ECs with NO consequently suppressed MMP-2 secretion revealed by zymographic assay. Functional analysis of MMP-2 promoter (1716 bps) indicated that p53-binding site (-1659 to -1629) was crucial for MMP-2 promoter activity. ATF3 has been reported to act as a transcriptional repressor for p53. ECs treated with NO induced ATF3 expression. Consistently, Ad-eNOS-infected ECs showed an increase of ATF3 level. Moreover, ECs either over-expressed ATF3 or treated with an ATF3 activator (MG-132) resulted in a repression of MMP-2 promoter activity. As a result of MMP-2 suppression by NO, ECs treated with NO inhibited endothelial migration, a phenomenon similar to ECs treated with MMP-2 antibody or MG-132. These results indicate that the attenuation of endothelial migration by NO is mediated at least in part by its reduction of MMP-2 expression via the up-regulation of ATF3. This study provides a molecular basis which supports that NO acts as a negative regulator via induction of ATF3 to suppress MMP-2 gene expression in endothelial migration.
15d-PGJ2 derived from arachidonic acid is able to activate peroxisome proliferator-activated receptor γ(PPAR-γ), one of the nuclear hormone receptor superfamily. PPAR-γ expression in ECs, macrophages, and vascular smooth muscle cells (SMCs) has been found in the atherosclerotic lesion.15d-PGJ2 has been shown to induce NO production and ATF3 expression and inhibit cell migration. Present study further demonstrated that a time-dependent inhibition of MMP-2 expression in ECs treated with 15d-PGJ2.The inhibitory effect of 15d-PGJ2 on MMP-2 expression was a transcriptional event since 15d-PGJ2 reduced MMP-2 promoter activity and the MMP-2 mRNA level was unaffected in ECs pre-treated with actinomycin D.15d-PGJ2 treatment of ECs consistently suppressed MMP-2 secretion revealed by zymographic assay. Moreover, 15d-PGJ2 treatment increased NO release due to eNOS activation and this was inhibited by pre-treating ECs with L-NAME.15d-PGJ2 increased the ATF3 protein level in a dose- and time-dependent manner. The 15d-PGJ2-induced ATF3 expression in ECs is mediated via p38 signaling pathway since ATF3 expression was inhibited by either treatment with P38 inhibitor (SB203580) or transient transfection with dominant negative mutant of p38. These results demonstrate that the p38 signaling pathway and the increased NO production are involved in the inhibitory effect of 15d-PGJ2 on MMP-2 via an induction of ATF3.
IL-1b has been implicated to contribute to atherosclerosis. Precious studies have demonstrated that IL-1b treatment increases the production of ROS in ECs and SMCs. Whether, the ROS production is involved in MMP-2 expression in IL-1b-stimulated ECs is unclear. Present study demonstrated that the ATF3 expression in ECs was induced by IL-1b with a dose- and time-dependant manner. ECs treated with IL-1b increased intracellular ROS. Pretreated ECs with N-acetyl-L-cysteine (NAC) blocked IL-1b-induced ATF3 expression, indicating that ROS was involved in the ATF3 induction by IL-1b. The Rac1 pull-down assay demonstrated that Rac1 activation in ECs treated with IL-1b required ROS. Transiently transfected ECs with the dominant negative mutant of Rac1 (RacN17) suppressed the IL-1b-induced ATF3. Moreover, ECs transfected with the dominant positive mutant of Rac1 (RacV12) increased ATF3 expression. Thus, Rac1 activation by ROS is involved in the IL-1b-induced ATF3 expression. Induction of ATF3 was shown after over-expression of MEKK-1, a JNK kinase, in ECs. ECs pretreated with curcumin and SP600126 but not of SB203580 or PD98059 inhibited IL-1b-induced ATF3 expression. Thus, JNK pathway activation is a major signaling pathway leading to IL-1b-induced ATF3 expression. The IL-1b-induced ATF3 expression resulted in a suppression of MMP-2 secretion with a time-dependent manner. Our results suggest that ATF3 expression is mediated via the increase of ROS production and Rac1 activation that lead to JNK activation. The IL-1b-induced ATF3 expression results in a suppression of MMP-2 expression.
Taken together, these results indicate that there is a common suppression mechanism of MMP-2 expression in which ATF3 induction plays a role in ECs under oxidative stress. This study also provides a molecular basis that supports the notion that redox imbalance plays an importance role during vascular dysfunction.

頁次
正文目錄…………………………………………………………………………………………………………………………………I
附圖目錄…………………………………………………………………………………………………………………………………IV
關鍵詞……………………………………………………………………………………………………………………………………VII
中文摘要………………………………………………………………………………………………………………………………XII
英文摘要……………………………………………………………………………………………………………………………XVII
第一章 緒言……………………………………………………………………………………………………………………………1
第一節 研究動機與目的…………………………………………………………………………………………1
第二節 研究文獻探討………………………………………………………………………………………………4
第二章 研究材料與方法………………………………………………………………………………………………23
第一節 實驗材料與儀器……………………………………………………………………………………….23
一﹑藥品……………………………………………………………………………………………………………………………23
二﹑材料……………………………………………………………………………………………………………………………25
三﹑細胞培養與分析測定使用之溶液……………………………………………………………28
四﹑細胞種類…………………………………………………………………………………………………………………33
五﹑儀器設備…………………………………………………………………………………………………………………34
第二節 實驗方法……………………………………………………………………………………………………….37
一、 細胞培養………………………………………………………………………………………………………………37
二、 cDNA 探針與啟動子合成、製作與純化………………………………………………39
三、 DNA碎裂與細胞存活測定……………………………………………………………………………41
四、 一氧化氮測定……………………………………………………………………………………………………43
五、 北方墨點轉印法………………………………………………………………………………………43
六、 Zymographic測定……………………………………………………………………………………………46
七、 腺病毒帶eNOS基因質體感染………………………………………………………………47
八、 基因質體轉染與發光酶測定…………………………………………………………………….47
九、 活體外內皮細胞的遷移測定……………………………………………………………………48
十、 細胞增生測法……………………………………………………………………………………………………48
十一、 西方墨點轉印法……………………………………………………………………………….49
十二、 活細胞內ROS含量測定……………………………………………………………………….51
十三、 Rac1 下式活性測定………………………………………………………………………………51
第三章 結果…………………………………………………………………………………………………………………………54
第一節 一氧化氮對內皮細胞中MMP-2之表現:活化轉錄因子3 (ATF3) 所扮演之角色……………………………………………………………………….54
第二節 15-deoxy-D12,14-PGJ2 對內皮細胞中MMP-2之表現:活化轉錄因子3 (ATF3) 所扮演之角色…………………………………………………71
第三節 介白素-1b 對內皮細胞中MMP-2之表現:活化轉錄因子3 (ATF3) 所扮演之角色……………………………………………………………………….85
第四章 討論與結論………………………………………………………………………………………………………103
第一節 討論……………………………………………………………………………………………………………103
第二節 結論……………………………………………………………………………………………………………113
參考文獻………………………………………………………………………………………………………………………………116
附錄…………………………………………………………………………………………………………………………………………127

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