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系統識別號 U0026-0812200911300661
論文名稱(中文) 凝血酶調節素在血管平滑肌細胞遷移與血管修復的角色
論文名稱(英文) The Role of Thrombomodulin in Vascular Smooth Muscle Cell Migration and Vascular Remodeling
校院名稱 成功大學
系所名稱(中) 生物化學暨分子生物學研究所
系所名稱(英) of Biochemistry and Molecular Biology
學年度 93
學期 2
出版年 94
研究生(中文) 徐孟瑜
研究生(英文) Meng-Yu Hsu
學號 s1692103
學位類別 碩士
語文別 中文
論文頁數 121頁
口試委員 指導教授-施桂月
口試委員-吳華林
口試委員-林淑華
口試委員-江美治
中文關鍵字 凝血酶調節素  平滑肌細胞  血管修復 
英文關鍵字 Thrombomodulin  smooth muscle cell  remodeling 
學科別分類
中文摘要 動脈粥狀硬化是心血管疾病中主要疾病,然而在動脈粥狀病變與血管再窄化發展病程中,血管平滑肌細胞從中膜層遷移至新生血管內膜是血管形成病變的重要的因素。 人類凝血酶調節素 (thrombomodulin,TM)首先被發現於血管內皮細胞表面上,其主要的生理功能為維持血液循環系統的抗凝性。然而凝血酶調節素也表現在病患血管平滑肌細胞,以及泡沫狀巨噬細胞上。並且凝血酶調節素能夠調節在靜脈移植後形成的動脈粥狀硬化,與血管再窄化中病理學上的改變。然而凝血酶調節素是經由何種機制調控此現象目前仍未有很清楚的相關研究報告。凝血酶調節素為細胞表面的穿膜醣蛋白,全長為557個胺基酸,在結構上具有五個功能區,依序為似lectin功能區 (TMD1)、六個縱列排列的表皮生長因子調節區(TMD2)、絲胺酸/息寧胺酸胺基酸區域(TMD3)、穿膜功能區(TMD4)與細胞質功能區(TMD5)。在細胞遷移實驗中,我們證實凝血酶調節素重組蛋白(TMD23)能夠促進血管平滑肌細胞遷移的能力,並且抑制 αvβ3 integrin 能夠顯著的抑制由TMD23引發平滑肌細胞遷移的現象。當細胞先以 U0126 (ERK1/2抑制劑 )、P38 (SB203580抑制劑) 、 LY294002( PI3K/AKT 抑制劑 )處理均能夠有效的抑制細胞遷移的現象。然而細胞遷移能力並不會受到 PKA 抑制劑 KT5720 的影響。在動物實驗中,利用攜帶有凝血酶調節素重組蛋白的微滲透囊,經由皮下植入在血管綁結手術後的小鼠背部,實驗結果顯示,內膜層除上中膜層的比值(I/M ratio),mTMD23 (0.4+/-0.17, n=7) 、mTMD123 (0.4+/-0.35, n=4) 與 PBS對照組(1.45+/-0.6, n=12)相比,可減緩血管再塑 remodeling 的情形達三倍之多,而 mTMD1 (0.63+/-0.51, n=7) 亦達兩倍之多,顯示凝血酶調節素具有減緩血管窄化的能力。在 ApoE 基因剔除小鼠實驗中,我們也證實處理 mTMD1與 mTMD23 能夠降低動脈粥狀硬化的病變。

英文摘要 Atherosclerosis is the underlying disorder in the majority of patients with cardiovascular diseases. The migration of vascular smooth muscle cells (VSMCs) from the media into the neointima is a key event in the development of atherosclerotic lesion and in restenosis. Thrombomodulin (TM) is an endothelial membrane anticoagulant protein that has previously been recognized in endothelial cells. However, TM is expressed in intimal vascular smooth muscle cells (VSMCs) and foamy macrophages in atherosclerotic region. TM also modulated pathological alterations of the vessel wall in restenosis or vein graft atherosclerosis. However, the mechanisms involved in this process have not been fully characterized. TM is a glycosylated type І transmembrane molecule of 557 amino acids. It possesses a domain structure comprising a lectin-like domain (TMD1), six tandem epidermal growth factor-like domains (TMD2), a serine/threoine-rich domain (TMD3), a transmembrane domain (TMD4), and an intracellular domain (TMD5). In migration assay we demonstrated that recombinant TMD23 potently promoted VSMCs migration. Inhibition of αvβ3 integrin resulted in a significant decrease of SMC migration. Migration stimulated by TMD23 is significantly reduced by extracellular signal-regulated kinase 1/2 inhibitor, U0126, the p38 inhibitor, SB203580, or the phosphatidylinositol 3 kinase inhibitor, LY294002, but not PKA inhibitor, KT5720. In vivo assay, osmotic mini-pump containing mTMD1, mTMD23 or mTMD123 was implanted subcutaneously for 28 days while performing ligation experiment in male C57BL/6 mice. The intima/media ratio of the occluded vessels of control mice treated with PBS (1.45+/-0.6, n=12) is two-fold higher than that of TMD1-treated mice (0.63+/-0.51, n=7), three-fold higher than those of TMD23-treated mice (0.4+/-0.17, n=7) and TMD123-treated mice (0.4+/-0.35, n=4). The recombinatnt TM proteins significantly decreased neointima formation in ligation model. In the ApoE knockout model, we demonstrated that atherosclerotic plaques of mTMD1 and mTMD23-treated mice were significantly decreased than those in PBS control.

論文目次 中文摘要…………………………………………………………………… 3
英文摘要…………………………………………………………………… 5
誌謝………………………………………………………………………… 7
目錄………………………………………………………………………… 8
圖目錄……………………………………………………………………… 10
表目錄……………………………………………………………………… 12
縮寫檢索表………………………………………………………………… 13
儀器與藥品………………………………………………………………… 15
緒論………………………………………………………………………… 21
研究動機…………………………………………………………………… 30
材料與方法………………………………………………………………… 31
一、 重組凝血酶調節素基因的選殖技術…………………………………31
二、聚合酶連鎖反應(Polymerase Chain Reaction;PCR) …………………35
三、QuikChange Site-Directed Mutagenesis…………………………………37
四、酵母菌蛋白質表現與純化系統……………………………………… 39
五、 凝血酶調節素活性測試………………………………………………45
六、 細胞培養方法…………………………………………………………47
七、 細胞移動實驗 (cell migration assay) …………………………………48
八、 細胞附著實驗(Cell adhesion assay)……………………………… 50
九、 蛋白質基本技術操作…………………………………………………52
十、動物實驗……………………………………………………………… 57
十一、組織切片及染色…………………………………………………… 59
十二、血管檢查評估……………………………………………………… 61
結果………………………………………………………………………… 63
討論………………………………………………………………………… 69
參考文獻…………………………………………………………………… 76
圖…………………………………………………………………………… 84
表…………………………………………………………………………… 110
自述………………………………………………………………………… 122
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