急性缺氧下血管細胞反應之分子機轉

Taipei Medical University Institutional Repository > 醫學院 > 臨床醫學研究所 > 博碩士論文 > Item 987654321/4218

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题名:	急性缺氧下血管細胞反應之分子機轉 Mechanisms of Cellular and Molecular Responses to Acute Hypoxia in Vascular Cells
作者:	陳識中 Shih-Chung Chen
贡献者:	臨床醫學研究所
关键词:	活化轉錄因子3 盤狀結構域受體2 血管平滑肌細胞間質金屬蛋白酶 -2 硝基化蛋白質 discoidin domain receptor-2 vascular smooth mucle cell matrix metalloproteinase-2 activating transcription factor 3 S-nitrosylation
日期:	2009
上传时间:	2009-08-27 16:44:09 (UTC+8)
摘要:	本論文的主要目的為研究缺氧對於血管內皮與平滑肌細胞的影響及其相關分子機制。盤狀結構域受體2 (discoidin domain receptor-2, DDR2) 是一種會與細胞間質結合的酪氨酸激酶 (tyrosin kinase)。本論文研究結果發現，在缺氧逆境下血管平滑肌細胞 (vascular smooth muscle cells, VSMCs) 中的DDR2 會增加表現。此外，處理p38 mitogen-activated protein kinase 的專一性抑制劑 (SB203580) 以及利用RNA 干擾技術都可以抑制缺氧逆境所誘導的DDR2 表現量。進一步進行啟動子的活性分析中，發現缺氧逆境可以增加Myc-Max-DNA 結合在DDR2 的啟動子上並造成膠體移位 (gel shift)。我們亦發現缺氧逆境可以增加血管平滑肌細胞的間質金屬蛋白酶 (matrix metalloproteinase-2, MMP-2) 活性進而加速其細胞的移動，而這些現象都會被DDR2 的抑制劑及干擾性RNA 予以抑制。因此，缺氧逆境可以增加DDR2 基因的表現並藉由p38MAPK 的途徑來加速血管平滑肌細胞的移動。活化轉錄因子3 (activating transcription factor 3, ATF3) 是一個非常重要的細胞逆境反應因子，在本論文中我們發現血管細胞在短暫的缺氧逆境下可以增加ATF3、c-jun-N(JNK)及內皮細胞一氧化氮合成酶(endothelial nitric oxide synthase, eNOS)的基因表現。處裡JNK 的專一性抑制劑會降低ATF3 的表現，然若處裡eNOS 的作用抑制劑 (LNAME)或PI-3 kinase 也會抑制ATF3 基因表現。因此短暫性的缺氧逆境下ATF3 應是透過一氧化氮或是JNK 途徑來活化，且細胞中的 MMP-2 基因的表現會受抑制。可見在不同細胞中缺氧逆境所造成的生理現象是不同的。近年來由於一氧化氮在細胞中扮演相當重要的角色，為了進一步探討在缺氧逆境下一氧化氮對細胞蛋白質的影響，我們利用改良式的biotin switch 方法並配合蛋白質體技術來進行硝基化 (S-nitrosylation) 蛋白質的篩選。經由串聯式質譜儀發現共有11 個蛋白質在缺氧逆境下會增加其半胱氨酸殘基的硝基化作用。這其中包含細胞骨架蛋白、控制雙硫鍵形成蛋白與基因調節蛋白。藉由這些硝基化蛋白質的發現並配合之前DDR2 與ATF3 等蛋白的研究，我們將可進一步釐清缺氧逆境在血管細胞中扮演的生理角色。 The aim of this thesis is to investigate the responses of vascular cells, including endothelial cells (ECs) and smooth muscle cells (VSMCs), to acute hypoxia and the underlying molecular mechanisms. Discoidin domain receptor-2 (DDR2) is a receptor tyrosine kinase that binds to the extracellular matrix. We first investigated the role of hypoxia in DDR2 expression in VSMCs and the underlying mechanism. Subjecting VSMCs to hypoxia induced DDR2 expression; treatments with a specific inhibitor (SB203580) of p38 mitogen-activated protein kinase (MAPK) or p38-specific small interference RNA (siRNA) abolished this hypoxia-induced DDR2 expression. Gel shifting assays showed that hypoxia increased the Myc–Max–DNA binding activity in the promoter region of DDR2; inhibition of p38 MAPK activation by SB203580 and p38-specific siRNA blocked hypoxia-induced DDR2 promoter activity. Hypoxia also induced matrix metalloproteinase-2 (MMP-2) activity in VSMCs and increased their migration. These VSMC responses to hypoxia were inhibited by DDR2- and p38-specific siRNAs. Our results suggested that hypoxia induces DDR2 expression in VSMCs at the transcriptional level, which is mediated by the p38 MAPK pathway and contributes to VSMC migration.Since activating transcription factor 3 (ATF3), a stress-inducible transcription factor playing significant roles in cellular responses to stress, including hypoxia, we further investigated the role of ATF3 in cellular responses to hypoxia. Vascular ECs were subjected to acute hypoxia and ATF3 expression was examined. ECs exposed to hypoxia transiently induced ATF3 expression. A transient increase in the activation of c-Jun-NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) in ECs was observed; however, only ECs pretreated with a specific inhibitor to JNK suppressed the hypoxia-induced ATF3 expression. ECs exposed to acute hypoxia transiently increased endothelial nitric oxide synthase (eNOS) activity. Pre-treating ECs with a specific inhibitor to eNOS (L-NAME) or PI3-kinase significantly inhibited the hypoxia-induced JNK activation and ATF3 expression. ATF3 induction has been shown to inhibit matrix metalloproteinase-2 (MMP-2) expression. Consistently, ECs exposed to hypoxia attenuated the MMP-2 expression. These results suggest that the ATF3 induction by acute hypoxia is mediated by nitric oxide and the JNK pathway in ECs. Recent studies indicated that protein S-nitrosylation (S-NO) plays an important role in regulating proteins’ function and thus is of significance in cell physiology. We further investigated the effect of hypoxia on EC S-NO. A modified biotin-switch method coupled with proteomic approach demonstrated that at least 11 major proteins have significant increase in S-NO in ECs after acute hypoxia. Mass analysis by CapLC/Q-TOF identified those as Ras-GTPase-activating protein, protein disulfide-isomerase, human elongation factor-1-delta, tyrosine 3/tryptophan 5-monooxygenase activating protein, and several cytoskeleton proteins. Further understanding of the functional relevance of these S-nitrosylated proteins and the previous findings of DDR2 and ATF3 activities may provide a molecular basis for understanding the ischemia-induced vascular physiologies.
数据类型:	thesis
显示于类别:	[臨床醫學研究所] 博碩士論文

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