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系統識別號 U0026-0902201115592600
論文名稱(中文) 凝血酶調節素在血管平滑肌細胞的移行與發炎反應所扮演的角色
論文名稱(英文) The role of thrombomodulin in the migration and inflammatory responses of vascular smooth muscle cells
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 99
學期 1
出版年 100
研究生(中文) 白濬芃
研究生(英文) Chun-Peng Pai
學號 t9697104
學位類別 碩士
語文別 英文
論文頁數 63頁
口試委員 指導教授-江美治
口試委員-吳佳慶
口試委員-吳華林
口試委員-鄭宏祺
中文關鍵字 凝血酶調節素  血管平滑肌細胞  移行  發炎反應 
英文關鍵字 thrombomodulin  vascular smooth muscle cell  migration  inflammatory response 
學科別分類
中文摘要 血管平滑肌細胞的移行和增生在動脈粥狀硬化形成的過程中扮演著重要的角色。凝血酶調節素 (Thrombomodulin; TM) 是一個抗凝血蛋白,並有多種其他功能,包含抗發炎、促進細胞-細胞間黏附和促進血管新生。在健康的動脈的平滑肌細胞中,無法偵測到凝血酶調節素的表現;但是在動脈粥狀硬化和受傷所誘發增生的血管內層,血管平滑肌細胞中凝血酶調節素的表現量明顯增加。凝血酶調節素在血管平滑肌細胞的功能仍然不清楚。我們探討凝血酶調節素在血管平滑肌細胞的移行、增生和發炎反應中所扮演的角色。我們利用一株不表現凝血酶調節素訊息核糖核酸 (messenger RNA) 的胎鼠主動脈平滑肌細胞,A7r5,來探討過量表現凝血酶調節素對平滑肌細胞功能的影響。在過量表現帶有綠色螢光蛋白的凝血酶調節素 (TMGFP, 簡稱TMG) 的A7r5細胞,其由脂多醣 (lipopolysaccharide;LPS) 所誘發的單核球趨化蛋白-1 (monocyte chemoattractant protein-1) 和間白素-1α (interleukin-1α) 的訊息核醣核酸表現較過量表現綠色螢光蛋白的細胞有降低的趨勢。可是過量表現TMG和去除凝集素相似區域的凝血酶調節素 (lectin-like domain-deleted TMG;TMG∆L)對由脂多醣引起的甲型腫瘤壞死因子(tumor necrosis factor-α)、誘導型一氧化氮合成酵素 (inducible nitric oxide synthase) 和第一型細胞間黏附分子 (intercellular adhesion molecule-1) 的訊息核醣核酸表現沒有影響。過量表現TMG也不影響由血小板衍生性生長因子-BB (platelet-derived growth factor-BB;PDGF-BB) 所刺激的A7r5細胞的增生。在人類主動脈平滑肌細胞中,降低凝血酶調節素顯著地抑制由PDGF-BB刺激的細胞移行。在A7r5細胞中,過量表現TMG的細胞其移動力較過量表現GFP的細胞顯著增加。我也探討兩個凝血酶調節素突變蛋白,TMG∆L與TMS490,492AG,對細胞移動力的作用。TMS490,492AG因突變而喪失軟骨硫素 (chondroitin sulfate) 對凝血酶調節素的修飾作用。結果顯示,在第490與492胺基酸由絲胺酸 (serine) 變成丙胺酸 (alanine) 的凝血酶調節素 (TMS490,492AG) 失去增進細胞移動力的效果,而去除凝集素相似區域 (TMG∆L)則無影響。穩定表現TMG的A7r5細胞株,其在第一型膠原蛋白 (Type I collagen)覆蓋的表面的黏著能力,較表現GFP的細胞株為佳。在相同的條件下,TMG的表現亦有促進黏著斑激酶 (focal adhesion kinase) 磷酸化的作用。以上結果顯示,凝血酶調節素參與由PDGF-BB所刺激的血管平滑肌細胞的移行。凝血酶調節素可能透過軟骨硫素的作用來增加細胞與胞外間質的黏著性和黏著斑激酶的磷酸化,因而促進細胞的移行。
英文摘要 The migration and proliferation of vascular smooth muscle cells (VSMC) play important roles in atherosclerosis progression. Thrombomodulin (TM) is an anticoagulant protein with multiple additional functions, including anti-inflammation, enhancing cell-cell adhesion, and pro-angiogenesis. TM expression is not detected in VSMC of healthy arteries but is upregulated in VSMC of atherosclerotic vessels and during injury-induced neointima formation. The functional roles of TM expressed in VSMC remain unclear. We examined the role of TM in the migration, proliferation and inflammatory responses of VSMC. The effects of TM overexpression on VSMC activities were examined in A7r5 cells, an embryonic rat aortic VSMC cell line that does not express TM mRNA. A7r5 cells overexpressing a gene construct containing TM and green fluorescence protein (TMGFP, abbreviated as TMG) exhibited the trend of decreasing lipopolysaccharide (LPS)-induced mRNA expression of monocyte chemoattrant protein-1 and interleukin-1α compared to GFP-overexpressing cells. However, overexpression of TMG and its lectin-like domain-deleted mutant (TMG∆L) had no effect on LPS-induced mRNA expression of tumor necrosis factor-α, inducible nitric oxide synthase and intercellular adhesion molecule-1. Overexpression of TMG also had no effect on the proliferation of A7r5 cells stimulated with platelet-derived growth factor-BB (PDGF-BB). In human aortic VSMC, TM knockdown markedly inhibited PDGF-BB-stimulated migration. In A7r5 cells, overexpression of TMG increased cell motility compared to GFP overexpression. The effects of expressing two TM mutants, TMG∆L and TMS490,492AG (which blocked the chondroitin sulfate modification of TM), were also examined. The mutation of S490,492A eliminated the stimulatory effect of TM on cell motility whereas the deletion of the lectin-like domain had no effect. A7r5 cells stably expressing TMG exhibited greater ability of adhesion to type I collagen-coated surface compared to the GFP-expressing cells. Furthermore, TMG expression enhanced focal adhesion kinase (FAK) phosphorylation under the same condition. These results suggest that TM participates in PDGF-BB-stimulated VSMC migration through chondroitin sulfate moiety-mediated increases in cell adhesion and FAK phosphorylation.
論文目次 摘要 iii
Abstract v
1. Introduction 1
1.1. Thrombomodulin structure 1
1.2. The anti-coagulant function of thrombomodulin 3
1.3. Thrombomodulin in cardiovascular diseases. 4
1.4. Thrombomodulin in inflammation 6
1.5. Thrombomodulin in cell adhesion 8
1.6. TM in cell migration 9
3. Materials and Methods 11
3.1. Materials 11
3.1.1. Chemicals 11
3.1.2. Solution preparation 15
3.2. Methods 22
3.2.1. Cell Culture 22
3.2.2. Western blotting 23
3.2.3. RNA extraction and Reverse transcriptase-polymerase chain reaction (RT-PCR) 27
3.2.4. Lentivirus amplification and infection 30
3.2.6. Ki67 immunofluorescence staining 31
3.2.7. Migration assay 32
3.2.8. Detection of TM localization by confocol microscopy 33
3.2.9. Collagen matrix preparation 34
3.2.10. Statistical analysis 34
4. Results 35
4.1. TM localization in VSMCs. 35
4.2. TM did not significantly decrease LPS-induced mRNA expression of pro-inflammatory molecules in VSMC. 35
4.3. TM did not affect PDGF-induced VSMC proliferation. 36
4.4. TM knockdown decreased PDGF-induced VSMC migration. 37
4.5. TM overexpression increased PDGF-induced VSMC migration and TMS490, 492A mutation eliminated this effect. 37
4.6. TM overexpression increased cell adhesion and FAK phosphorylation in A7r5 cells on type I collagen-coated surface. 38
5. Discussion 39
6. References 44
7. Figures 56

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