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系統識別號 U0026-0812200913371975
論文名稱(中文) 小鼠頸動脈結紮模式中血管平滑肌細胞型態轉變過程之研究
論文名稱(英文) A time course study of smooth muscle cell phenotypic changes in mouse carotid artery ligation model
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 95
學期 2
出版年 96
研究生(中文) 周凌慧
研究生(英文) Ling-hui Chou
電子信箱 jasmine680705@yahoo.com.tw
學號 t9693105
學位類別 碩士
語文別 中文
論文頁數 65頁
口試委員 口試委員-李貽恆
指導教授-江美治
口試委員-吳梨華
口試委員-陳麗玉
中文關鍵字 新生內膜  平滑肌細胞  頸動脈結紮 
英文關鍵字 neointima  carotid artery ligation  smooth muscle cells 
學科別分類
中文摘要 在血管受傷病變過程中,平滑肌細胞會由收縮態轉變為合成態,並由血管中層往內膜層移行和增生,導致血管內膜增厚。平滑肌分化指標蛋白表現量的變化被當成是平滑肌型態轉變的一個表徵。先前的研究發現,在氣球擴張術所導致的血管重組過程中,血管中層的平滑肌細胞會由原先的收縮態轉變成合成態,之後再回復到收縮態。凝血酶調節素(Thrombomodulin; TM)是大量表現在血管內皮細胞表面的醣蛋白。TM與抗凝血、細胞與細胞之間的黏著以及抗發炎有關,並且扮演著重要角色。之前的研究顯示,在頸動脈結紮所誘發的新生內膜層以及動脈粥狀硬化血管的平滑肌細胞中,TM均會表現。在培養的平滑肌細胞,血小板源生性生長因子(PDGF-BB)會促使平滑肌細胞表現合成態;此外,我們實驗室的研究發現, PDGF-BB的刺激,可顯著增加培養的平滑肌細胞中TM的表現量。因此,本實驗的主要目的是利用血管結紮所導致的血管重組的模式,探討平滑肌細胞表現型與TM表現之間的相關性。本實驗採用八週大的C57BL/6公鼠,共分為四組,分別為:一、結紮後一週;二、結紮後兩週;三、結紮後四週;四、結紮後八週;五、對照組,採用左側未結紮總的頸動脈當作對照組。利用H&E染色方法分析血管壁截面積的變化。結果顯示,在結紮後一週及兩週,四週血管中層的面積較對照組明顯增加。在結紮後兩週及四週血管新生內膜層的面積均大幅增加,並在結紮後八週達到最大。血管中層的細胞數目,在結紮後兩週及四週較對照組明顯增加。而血管新生內膜層的細胞數在結紮後兩週至八週較對照組皆有明顯增加。此外,血管平滑肌分化指標蛋白sm-α-actin 和 sm-myosin heavy chain (sm-MHC)的表現量,在結紮後兩週、四週及八週皆顯著減少,且sm-MHC的變化比sm-α-actin更顯著。相對地,nm-MHC的表現量在結紮後一週即較對照組增加,到了結紮後兩週、四週及八週,其增加量更為顯著。我們進一步利用細胞增殖標記(5-bromo-2’-deoxyuridine;BrdU)來探討sm-MHC的表現與細胞增生的相關性,結果顯示,在結紮後兩週,血管新生內膜層的細胞增生比率達到最高,且增生的細胞與表現sm-MHC的細胞沒有重疊。我們另外探討PDGF-BB在頸動脈結紮後的變化,結果顯示,血管中層PDGF-BB的表現量在結紮後ㄧ週、兩週或四週較對照組明顯增加;血管新生內膜層的PDGF-BB的表現量,在結紮後的兩週及四週均大幅增加。最後,我們利用連續切片來探討TM與PDGF-BB表現的相關性。結果顯示,在結紮後一週至八週,血管TM及PDGF-BB的表現量均明顯增加,且兩者表現的模式類似。以上的結果顯示,在小鼠頸動脈結紮後所導致的血管重組過程中,平滑肌細胞型態會由原先的收縮態轉變成合成態,直到結紮後八週,平滑肌細胞尚未回復成收縮態。然而,當平滑肌分化指標蛋白表現量減少時,PDGF-BB、TM與nmMHC表現量皆增加,顯示在血管重組過程中,TM可能為平滑肌細胞去分化的指標。
英文摘要 An important hallmark of vascular injury is the increased proliferation and migration of vascular smooth muscle cells (VSMCs) associated with the transformation from contractile to synthetic phenotype. The phenotypic change of VSMCs is characterized by altered expression levels of differentiation markers. Previous studies showed that in media VSMCs undergo phenotypic transition first from contractile to synthetic phenotype, then back to contractile phenotype after balloon angioplasty-induced injury. Thrombomodulin (TM), a glycoprotein abundantly expressed on the endothelial cell surface, plays important roles in anti-coagulation, cell-cell adhesion and anti-inflammation. It was previously reported that TM was expressed in VSMCs of atherosclerotic arteries and during neointimal formation following carotid artery (CA) ligation. In vitro studies showed that PDGF-BB promotes the expression of synthetic phenotype in VSMCs and results from our lab indicate that PDGF-BB is a potent inducer for TM expression in VSMCs. Therefore, this study was aimed to examine the expression pattern of TM and SMCs phenotype during arterial remodeling using carotid artery ligation as a model. Right common CA of eight weeks-old male C57BL/6 mice were ligated for 1, 2, 4 and 8 weeks. The medial area increased at 1, 2, and 4 weeks after ligation compared with control. Neointimal area substantially increased at 2 and 4 weeks after ligation, and reached maximum at 8 weeks after ligation. Cell number in the media increased at 2 and 4 weeks, whereas cell number in neointima significantly increased from 2 to 8 weeks after ligation. The expression of two SMC differentiation markers, smooth muscle-specific α-actin (sm-α-actin) and sm-myosin heavy chain (sm-MHC), decreased at 2, 4 and 8 weeks after ligation with the expression of sm-MHC decreasing more significantly than that of sm-α-actin. By contrast, the expression of nm-MHC increased at 1, 2, 4 and 8 weeks after ligation. The cell proliferation detected by BrdU incorporation mainly increased in neointima at 2 weeks after ligation, and was not detected in VSMCs expressing sm-MHC. In addition, the expression of PDGF-BB in media increased at 1, 2 and 4 weeks, and markedly increased in neointima at 2 and 4 weeks after ligation. Finally, the expression of PDGF-BB and TM both increased from 1 week to 8 weeks after ligation and exhibited similar expression pattern. These results demonstrated that following CA ligation VSMCs switch from contractile to synthetic phenotype and do not revert to contractile phenotype up to 8 weeks after ligation. While SMC differentiation markers are down-regulated, PDGF-BB and TM expression is upregulated along with nm-MHC, suggesting that TM may serve as a de-differentiation marker for VSMCs during vascular remodeling.
論文目次 英文摘要-------------------------------------------------- I
中文摘要--------------------------------------------------III
圖目錄-----------------------------------------------------V
緒論---------------------------------------------------------1
研究動機-------------------------------------------------- 13
材料與方法----------------------------------------------- 14
藥品儀器-------------------------------------------------- 23
結果-------------------------------------------------------- 27
討論-------------------------------------------------------- 38
圖表-------------------------------------------------------- 43
參考文獻-------------------------------------------------- 60
附圖-------------------------------------------------------- 65
參考文獻 1.Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arteriosclerosis, thrombosis, and vascular biology. 2003;23(2):168-175.
2.Bostrom H, Willetts K, Pekny M, Leveen P, Lindahl P, Hedstrand H, Pekna M, Hellstrom M, Gebre-Medhin S, Schalling M, Nilsson M, Kurland S, Tornell J, Heath JK, Betsholtz C. PDGF-A signaling is a critical event in lung alveolar myofibroblast development and alveogenesis. Cell. 1996;85(6):863-873
3.Clempus RE, Sorescu D, Dikalova AE, Pounkova L, Jo P, Sorescu GP, Schmidt HH, Lassegue B, Griendling KK. Nox4 is required for maintenance of the differentiated vascular smooth muscle cell phenotype. Arteriosclerosis, thrombosis, and vascular biology. 2007;27(1):42-48.
4.Conway EM, Van de Wouwer M, Pollefeyt S, Jurk K, Van Aken H, De Vriese A, Weitz JI, Weiler H, Hellings PW, Schaeffer P, Herbert JM, Collen D, Theilmeier G. The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. The Journal of experimental medicine. 2002;196(5):565-577.
5.Corjay MH, Thompson MM, Lynch KR, Owens GK. Differential effect of platelet-derived growth factor- versus serum-induced growth on smooth muscle alpha-actin and nonmuscle beta-actin mRNA expression in cultured rat aortic smooth muscle cells. The Journal of biological chemistry. 1989;264(18):10501-10506.
6.Daniel TO, Gibbs VC, Milfay DF, Garovoy MR, Williams LT. Thrombin stimulates c-sis gene expression in microvascular endothelial cells. The Journal of biological chemistry. 1986;261(21):9579-9582.
7.De Leon H, Scott NA, Martin F, Simonet L, Bernstein KE, Wilcox JN. Expression of nonmuscle myosin heavy chain-B isoform in the vessel wall of porcine coronary arteries after balloon angioplasty. Circulation research. 1997;80(4):514-519.
8.Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. The Journal of biological chemistry. 1989;264(9):4743-4746.
9.Garrels JI, Gibson W. Identification and characterization of multiple forms of actin. Cell. 1976;9(4 PT 2):793-805.
10.Heldin CH, Westermark B. Mechanism of action and in vivo role of platelet-derived growth factor. Physiological reviews. 1999;79(4):1283-1316.
11.Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. Role of PDGF-B and PDGFR-beta in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development (Cambridge, England). 1999;126(14):3047-3055.
12.Holycross BJ, Blank RS, Thompson MM, Peach MJ, Owens GK. Platelet-derived growth factor-BB-induced suppression of smooth muscle cell differentiation. Circulation research. 1992;71(6):1525-1532.
13.Huang HC, Shi GY, Jiang SJ, Shi CS, Wu CM, Yang HY, Wu HL. Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. The Journal of biological chemistry. 2003;278(47):46750-46759.
14.Jawien A, Bowen-Pope DF, Lindner V, Schwartz SM, Clowes AW. Platelet-derived growth factor promotes smooth muscle migration and intimal thickening in a rat model of balloon angioplasty. The Journal of clinical investigation. 1992;89(2):507-511.
15.Kourembanas S, Morita T, Liu Y, Christou H. Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Kidney international. 1997;51(2):438-443.
16.Kumar A, Lindner V. Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow. Arteriosclerosis, thrombosis, and vascular biology. 1997;17(10):2238-2244.
17.Kuro-o M, Nagai R, Tsuchimochi H, Katoh H, Yazaki Y, Ohkubo A, Takaku F. Developmentally regulated expression of vascular smooth muscle myosin heavy chain isoforms. The Journal of biological chemistry. 1989;264(31):18272-18275.
18.Levine GN, Chodos AP, Loscalzo J. Restenosis following coronary angioplasty: clinical presentations and therapeutic options. Clinical cardiology. 1995;18(12):693-703.
19.Li JM, Singh MJ, Itani M, Vasiliu C, Hendricks G, Baker SP, Hale JE, Rohrer MJ, Cutler BS, Nelson PR. Recombinant human thrombomodulin inhibits arterial neointimal hyperplasia after balloon injury. J Vasc Surg. 2004;39(5):1074-1083.
20.Li YH, Liu SL, Shi GY, Tseng GH, Liu PY, Wu HL. Thrombomodulin plays an important role in arterial remodeling and neointima formation in mouse carotid ligation model. Thrombosis and haemostasis. 2006;95(1):128-133.
21.Libby P, Warner SJ, Salomon RN, Birinyi LK. Production of platelet-derived growth factor-like mitogen by smooth-muscle cells from human atheroma. The New England journal of medicine. 1988;318(23):1493-1498.
22.Lindner V, Fingerle J, Reidy MA. Mouse model of arterial injury. Circulation research. 1993;73(5):792-796.
23.Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circulation research. 1994;75(5):803-812.
24.Mosca L, Manson JE, Sutherland SE, Langer RD, Manolio T, Barrett-Connor E. Cardiovascular disease in women: a statement for healthcare professionals from the American Heart Association. Writing Group. Circulation. 1997;96(7):2468-2482.
25.Owens GK. Regulation of differentiation of vascular smooth muscle cells. Physiological reviews. 1995;75(3):487-517.
26.Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiological reviews. 2004;84(3):767-801.
27.Owens GK, Thompson MM. Developmental changes in isoactin expression in rat aortic smooth muscle cells in vivo. Relationship between growth and cytodifferentiation. The Journal of biological chemistry. 1986;261(28):13373-13380.
28.Rabausch K, Bretschneider E, Sarbia M, Meyer-Kirchrath J, Censarek P, Pape R, Fischer JW, Schror K, Weber AA. Regulation of thrombomodulin expression in human vascular smooth muscle cells by COX-2-derived prostaglandins. Circulation research. 2005;96(1):e1-6.
29.Raines EW. PDGF and cardiovascular disease. Cytokine & growth factor reviews. 2004;15(4):237-254.
30.Ritchie KJ, Zhang DE. ISG15: the immunological kin of ubiquitin. Semin Cell Dev Biol. 2004;15(2):237-246.
31.Ross R. Atherosclerosis--an inflammatory disease. The New England journal of medicine. 1999;340(2):115-126.
32.Rovner AS, Murphy RA, Owens GK. Expression of smooth muscle and nonmuscle myosin heavy chains in cultured vascular smooth muscle cells. The Journal of biological chemistry. 1986;261(31):14740-14745.
33.Ruzicka DL, Schwartz RJ. Sequential activation of alpha-actin genes during avian cardiogenesis: vascular smooth muscle alpha-actin gene transcripts mark the onset of cardiomyocyte differentiation. The Journal of cell biology. 1988;107(6 Pt 2):2575-2586.
34.Schwartz SM, Campbell GR, Campbell JH. Replication of smooth muscle cells in vascular disease. Circulation research. 1986;58(4):427-444.
35.Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C, Huang HC, Chang YJ, Chen PS, Wu HL. Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation. 2005;111(13):1627-1636.
36.Stary HC, Blankenhorn DH, Chandler AB, Glagov S, Insull W, Jr., Richardson M, Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, et al. A definition of the intima of human arteries and of its atherosclerosis-prone regions. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arterioscler Thromb. 1992;12(1):120-134.
37.Stary HC, Chandler AB, Dinsmore RE, Fuster V, Glagov S, Insull W, Jr., Rosenfeld ME, Schwartz CJ, Wagner WD, Wissler RW. A definition of advanced types of atherosclerotic lesions and a histological classification of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Arteriosclerosis, thrombosis, and vascular biology. 1995;15(9):1512-1531.
38.Stary HC, Chandler AB, Glagov S, Guyton JR, Insull W, Jr., Rosenfeld ME, Schaffer SA, Schwartz CJ, Wagner WD, Wissler RW. A definition of initial, fatty streak, and intermediate lesions of atherosclerosis. A report from the Committee on Vascular Lesions of the Council on Arteriosclerosis, American Heart Association. Circulation. 1994;89(5):2462-2478.
39.Thyberg J. Phenotypic modulation of smooth muscle cells during formation of neointimal thickenings following vascular injury. Histology and histopathology. 1998;13(3):871-891.
40.Thyberg J, Blomgren K, Hedin U, Dryjski M. Phenotypic modulation of smooth muscle cells during the formation of neointimal thickenings in the rat carotid artery after balloon injury: an electron-microscopic and stereological study. Cell and tissue research. 1995;281(3):421-433.
41.Tohda G, Oida K, Okada Y, Kosaka S, Okada E, Takahashi S, Ishii H, Miyamori I. Expression of thrombomodulin in atherosclerotic lesions and mitogenic activity of recombinant thrombomodulin in vascular smooth muscle cells. Arteriosclerosis, thrombosis, and vascular biology. 1998;18(12):1861-1869.
42.Uchida K, Sasahara M, Morigami N, Hazama F, Kinoshita M. Expression of platelet-derived growth factor B-chain in neointimal smooth muscle cells of balloon injured rabbit femoral arteries. Atherosclerosis. 1996;124(1):9-23.
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