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系統識別號 U0026-2707201514344500
論文名稱(中文) 凝血酶調節素功能區域對於巨噬細胞極化現象之影響
論文名稱(英文) The effect of recombinant thrombomodulin domains in macrophage polarization
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) Department of Biochemistry and Molecular Biology
學年度 103
學期 2
出版年 104
研究生(中文) 王韻婷
研究生(英文) Yun-Ting Wang
學號 S16024092
學位類別 碩士
語文別 英文
論文頁數 74頁
口試委員 指導教授-吳華林
口試委員-施桂月
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  巨噬細胞極化現象  傷口癒合 
英文關鍵字 Thrombomodulin  Macrophage polarization  Wound healing 
學科別分類
中文摘要 巨噬細胞,受到輔助型T細胞的Th1和Th2細胞激素刺激之後,可以分別極化成經典型活化的M1型以及選擇性活化的M2型巨噬細胞。M1型的巨噬細胞可以促進吞噬作用以及發炎反應的發生;相反的,M2型巨噬細胞則會產生抗發炎細胞激素和促進血管新生因子幫助組織的修復。過去的報導指出,在糖尿病小鼠的傷口處會有持續性發炎反應以及增加M1型巨噬細胞數量,會延遲傷口癒合。因此巨噬細胞在傷口癒合的過程中,其功能以及型態上的轉變非常重要。凝血酶調節素(Thrombomodulin),屬於第一型的穿膜醣蛋白,其包含了五個功能區段,分別為類凝集素功能區段(C-type lectin-like domain)、類表皮生長因子功能區段 (EGF-like domain)、富含絲胺酸/蘇胺酸功能區段(serine/threonine-rich domain)、穿膜功能區段 (transmembrane domain)、細胞質功能區段 (cytoplasmic domain)。先前的研究提出,角質細胞表現TM並參與調控角質細胞的分化以及傷口癒合,但是目前為止TM的功能區域對於巨噬細胞極化現象的影響並不清楚。在此研究中,我們由老鼠腹腔分離出巨噬細胞,給予TMD23重組蛋白,經過培養,可以使細胞具有M2的型態。外加TMD23可以抑制γ-干擾素引起STAT1的磷酸化,並且促進介白素-4和介白素-10所引發STAT6和STAT3的活化。TMD23同時會藉由活化c-Myc轉錄因子促使M2巨噬細胞的標記,甘露醣受體的表現量增加。進一步的在活體實驗中,在老鼠傷口上給予重組蛋白TMD23會增加其傷口癒合的速度。TMD23可以藉由增加傷口處M2巨噬細胞的數量,降低傷口處發炎因子的含量、促進傷口的血管生成,幫助傷口癒合。綜合以上的結果,我們發現TMD23可以透過調控巨噬細胞的極化現象,以及抑制發炎反應加速傷口的癒合能力。
英文摘要 Macrophages can be induced to polarize to classically activated (M1) macrophages and alternatively activated (M2) macrophages by Th1 and Th2 cytokines, respectively. M1 macrophages promote phagocytosis and inflammatory response, whereas M2 macrophages can secret anti-inflammatory cytokines and angiogenic factors for tissue repair. It has been reported that persistent inflammation and increased M1 macrophages in wound area might contribute to the delayed wound healing in diabetic wounds, suggesting that functional and phenotypic switch of macrophages is a very important regulating process in cutaneous wound healing. Thrombomodulin (TM), a type I transmembrane glycoprotein, contains five functional domains, including a N-terminal C-type lectin-like domain, a six-repeated epidermal growth factor (EGF)-like domain, a serine/threonine-rich domain, a transmembrane domain, and a cytoplasmic domain. Previous research in our lab demonstrated that expression of TM in keratinocytes is involved in keratinocyte differentiation and wound healing. However, the function of recombinant TM domains in macrophage polarization remains unknown. In this study, we demonstrated that mouse peritoneal macrophages could be polarized to M2 phenotypes by treatment with recombinant EGF-like domain plus serine/threonine-rich domain of TM (rTMD23). The phosphorylation of signal transducer and activator of transcription (STAT) 1 induced by interferon- was inhibited by rTMD23, whereas the phosphorylation of STAT6 and STAT3 induced by interlukin-4 and interlukin-10 were enhanced by rTMD23. rTMD23 also enhanced CD206 expression, a marker for M2 phenotype, by increase of c-Myc expression in macrophages. Furthermore, local administration of rTMD23 to the cutaneous wound opening decreased the levels of inflammatory cytokines, promoted angiogenesis, increased the proportion of M2 macrophages in the wound area and accelerated wound closure in mice. In conclusion, we demonstrate that rTMD23 can accelerate wound healing by inducing M2 macrophage polarization and suppression of inflammatory response in wound opening.
論文目次 Chapter Page
Abstract in Chinese-----1
Abstract in English-----2
Acknowledgement---------3
Contents----------------4
List of figures---------5
Abbreviation------------6
Instruments-------------8
Reagents and Chemicals--10
Introduction------------14
Objective of this study-20
Materials and Methods---21
Results-----------------48
Conclusion--------------52
Discussion--------------53
References--------------56
Figures-----------------60
Appendixes--------------74


參考文獻 1.Sica A, Mantovani A: Macrophage plasticity and polarization: in vivo veritas. The Journal of clinical investigation 2012, 122(3):787-795.
2.Lawrence T, Natoli G: Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nature reviews Immunology 2011, 11(11):750-761.
3.Biswas SK, Mantovani A: Macrophage plasticity and interaction with lymphocyte subsets: cancer as a paradigm. Nature immunology 2010, 11(10):889-896.
4.Solinas G, Germano G, Mantovani A, Allavena P: Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation. Journal of leukocyte biology 2009, 86(5):1065-1073.
5.Sica A, Larghi P, Mancino A, Rubino L, Porta C, Totaro MG, Rimoldi M, Biswas SK, Allavena P, Mantovani A: Macrophage polarization in tumour progression. Seminars in cancer biology 2008, 18(5):349-355.
6.Moore KJ, Tabas I: Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145(3):341-355.
7.Moore KJ, Sheedy FJ, Fisher EA: Macrophages in atherosclerosis: a dynamic balance. Nature reviews Immunology 2013, 13(10):709-721.
8.Ferrante CJ, Leibovich SJ: Regulation of Macrophage Polarization and Wound Healing. Advances in wound care 2012, 1(1):10-16.
9. Dickhout JG, Basseri S, Austin RC: Macrophage function and its impact on atherosclerotic lesion composition, progression, and stability: the good, the bad, and the ugly. Arteriosclerosis, thrombosis, and vascular biology 2008, 28(8):1413-1415.
10.Pollard JW: Tumour-educated macrophages promote tumour progression and metastasis. Nature reviews Cancer 2004, 4(1):71-78.
11.Solinas G, Schiarea S, Liguori M, Fabbri M, Pesce S, Zammataro L, Pasqualini F, Nebuloni M, Chiabrando C, Mantovani A et al: Tumor-conditioned macrophages secrete migration-stimulating factor: a new marker for M2-polarization, influencing tumor cell motility. J Immunol 2010, 185(1):642-652.
12.Qian BZ, Pollard JW: Macrophage diversity enhances tumor progression and metastasis. Cell 2010, 141(1):39-51.
13.Hansson GK, Hermansson A: The immune system in atherosclerosis. Nature immunology 2011, 12(3):204-212.
14.Stoger JL, Goossens P, de Winther MP: Macrophage heterogeneity: relevance and functional implications in atherosclerosis. Current vascular pharmacology 2010, 8(2):233-248.
15.Wolfs IM, Donners MM, de Winther MP: Differentiation factors and cytokines in the atherosclerotic plaque micro-environment as a trigger for macrophage polarisation. Thrombosis and haemostasis 2011, 106(5):763-771.
16.Miao M, Niu Y, Xie T, Yuan B, Qing C, Lu S: Diabetes-impaired wound healing and altered macrophage activation: a possible pathophysiologic correlation. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society 2012, 20(2):203-213.
17.Singer AJ, Clark RA: Cutaneous wound healing. The New England journal of medicine 1999, 341(10):738-746.
18.Guo S, Dipietro LA: Factors affecting wound healing. Journal of dental research 2010, 89(3):219-229.
19.Gurtner GC, Werner S, Barrandon Y, Longaker MT: Wound repair and regeneration. Nature 2008, 453(7193):314-321.
20.Mahdavian Delavary B, van der Veer WM, van Egmond M, Niessen FB, Beelen RH: Macrophages in skin injury and repair. Immunobiology 2011, 216(7):753-762.
21.Daley JM, Brancato SK, Thomay AA, Reichner JS, Albina JE: The phenotype of murine wound macrophages. Journal of leukocyte biology 2010, 87(1):59-67.
22.Lucas T, Waisman A, Ranjan R, Roes J, Krieg T, Muller W, Roers A, Eming SA: Differential roles of macrophages in diverse phases of skin repair. J Immunol 2010, 184(7):3964-3977.
23.Campbell L, Saville CR, Murray PJ, Cruickshank SM, Hardman MJ: Local arginase 1 activity is required for cutaneous wound healing. The Journal of investigative dermatology 2013, 133(10):2461-2470.
24.Raife TJ, Lager DJ, Madison KC, Piette WW, Howard EJ, Sturm MT, Chen Y, Lentz SR: Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. The Journal of clinical investigation 1994, 93(4):1846-1851.
25.Suzuki K, Kusumoto H, Deyashiki Y, Nishioka J, Maruyama I, Zushi M, Kawahara S, Honda G, Yamamoto S, Horiguchi S: Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. The EMBO journal 1987, 6(7):1891-1897.
26.Healy AM, Rayburn HB, Rosenberg RD, Weiler H: Absence of the blood-clotting regulator thrombomodulin causes embryonic lethality in mice before development of a functional cardiovascular system. Proceedings of the National Academy of Sciences of the United States of America 1995, 92(3):850-854.
27.Conway EM, Van de Wouwer M, Pollefeyt S, Jurk K, Van Aken H, De Vriese A, Weitz JI, Weiler H, Hellings PW, Schaeffer P et al: 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.
28.Van de Wouwer M, Plaisance S, De Vriese A, Waelkens E, Collen D, Persson J, Daha MR, Conway EM: The lectin-like domain of thrombomodulin interferes with complement activation and protects against arthritis. Journal of thrombosis and haemostasis : JTH 2006, 4(8):1813-1824.
29.Abeyama K, Stern DM, Ito Y, Kawahara K, Yoshimoto Y, Tanaka M, Uchimura T, Ida N, Yamazaki Y, Yamada S et al: The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. The Journal of clinical investigation 2005, 115(5):1267-1274.
30.Shi CS, Shi GY, Hsiao HM, Kao YC, Kuo KL, Ma CY, Kuo CH, Chang BI, Chang CF, Lin CH et al: Lectin-like domain of thrombomodulin binds to its specific ligand Lewis Y antigen and neutralizes lipopolysaccharide-induced inflammatory response. Blood 2008, 112(9):3661-3670.
31.Ma CY, Chang WE, Shi GY, Chang BY, Cheng SE, Shih YT, Wu HL: Recombinant thrombomodulin inhibits lipopolysaccharide-induced inflammatory response by blocking the functions of CD14. J Immunol 2015, 194(4):1905-1915.
32.Little CD, Nau MM, Carney DN, Gazdar AF, Minna JD: Amplification and expression of the c-myc oncogene in human lung cancer cell lines. Nature 1983, 306(5939):194-196.
33.Mariani-Costantini R, Escot C, Theillet C, Gentile A, Merlo G, Lidereau R, Callahan R: In situ c-myc expression and genomic status of the c-myc locus in infiltrating ductal carcinomas of the breast. Cancer research 1988, 48(1):199-205.
34.Munzel P, Marx D, Kochel H, Schauer A, Bock KW: Genomic alterations of the c-myc protooncogene in relation to the overexpression of c-erbB2 and Ki-67 in human breast and cervix carcinomas. Journal of cancer research and clinical oncology 1991, 117(6):603-607.
35.Augenlicht LH, Wadler S, Corner G, Richards C, Ryan L, Multani AS, Pathak S, Benson A, Haller D, Heerdt BG: Low-level c-myc amplification in human colonic carcinoma cell lines and tumors: a frequent, p53-independent mutation associated with improved outcome in a randomized multi-institutional trial. Cancer research 1997, 57(9):1769-1775.
36. Dang CV: c-Myc target genes involved in cell growth, apoptosis, and metabolism. Molecular and cellular biology 1999, 19(1):1-11.
37.Davis AC, Wims M, Spotts GD, Hann SR, Bradley A: A null c-myc mutation causes lethality before 10.5 days of gestation in homozygotes and reduced fertility in heterozygous female mice. Genes & development 1993, 7(4):671-682.
38.Tsourdi E, Barthel A, Rietzsch H, Reichel A, Bornstein SR: Current aspects in the pathophysiology and treatment of chronic wounds in diabetes mellitus. BioMed research international 2013, 2013:385641.
39.Ziyadeh N, Fife D, Walker AM, Wilkinson GS, Seeger JD: A matched cohort study of the risk of cancer in users of becaplermin. Advances in skin & wound care 2011, 24(1):31-39.
40.Kampfer H, Schmidt R, Geisslinger G, Pfeilschifter J, Frank S: Wound inflammation in diabetic ob/ob mice: functional coupling of prostaglandin biosynthesis to cyclooxygenase-1 activity in diabetes-impaired wound healing. Diabetes 2005, 54(5):1543-1551.
41.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.
42.Wei HJ, Li YH, Shi GY, Liu SL, Chang PC, Kuo CH, Wu HL: Thrombomodulin domains attenuate atherosclerosis by inhibiting thrombin-induced endothelial cell activation. Cardiovascular research 2011, 92(2):317-327.
43.Pagel CN, Song SJ, Loh LH, Tudor EM, Murray-Rust TA, Pike RN, Mackie EJ: Thrombin-stimulated growth factor and cytokine expression in osteoblasts is mediated by protease-activated receptor-1 and prostanoids. Bone 2009, 44(5):813-821.
44.Li YH, Kuo CH, Shi GY, Wu HL: The role of thrombomodulin lectin-like domain in inflammation. Journal of biomedical science 2012, 19:34.
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