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系統識別號 U0026-2907201411302900
論文名稱(中文) 探討腫瘤內皮標誌1在皮膚傷口癒合中扮演的角色
論文名稱(英文) Study on the Role of Tumor Endothelial Marker 1 in Wound Healing
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 102
學期 2
出版年 103
研究生(中文) 李瑞庭
研究生(英文) Jui-Ting Li
學號 S16014089
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-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 腫瘤內皮標誌1  傷口癒合  纖維母細胞 
英文關鍵字 tumor endothelial marker 1  wound healing  fibroblasts 
學科別分類
中文摘要 腫瘤內皮標誌1(Tem1)又稱為內皮唾酸蛋白(Endosialin),與凝血酶調節素(TM)同屬C-type lectin-like domain蛋白家族,其結構包含六個結構區,分別是:類C型凝集結構區、sushi結構區、具有三次類表皮生長因子重複的結構區、類粘蛋白樣結構區、穿膜結構區、細胞膜內結構區。先前研究證實,小鼠皮膚在胚胎時期會大量表現Tem1,並在出生後降低表現量。但是,目前關於Tem1在皮膚生理功能的角色並不清楚。我們實驗室過去已經證實TM是參與調控皮膚上皮細胞分化及傷口癒合的重要蛋白質。因此,本研究的目的要探討Tem1於皮膚傷口癒合過程中所扮演的角色。小鼠皮膚纖維母細胞的傷口癒合實驗結果顯示,創傷刺激促使Tem1的基因表現量上升。轉化生長因子(TGF-β1)是傷口癒合過程中主要調控皮膚纖維母細胞基因表現的蛋白質,實驗亦發現TGF-β1能夠提升Tem1的基因表現,且是透過下游的胞外訊息調節激酶(ERK)磷酸化路徑。相反的,利用shRNA弱化皮膚纖維母細胞的Tem1基因表現則會干擾細胞貼附、爬行與增生,進而降低傷口癒合能力。進一步利用來自台灣大學林淑華老師實驗室的Tem1-lacZ 報導基因轉殖鼠,以了解Tem1在傷口癒合中表現的情況。結果發現,可在第五天傷口的真皮層偵測到Tem1的啟動子活性表現,且在第七、九天的傷口真皮層則有表現面積變大的現象。透過免疫組織化學染色法亦顯示,具有Tem1的啟動子活性的細胞,同時也表現活化態纖維母細胞標誌α-SMA。皮膚傷口癒合實驗中,Tem1基因剔除小鼠的癒合能力明顯較正常小鼠來的差。綜合以上結果,可推斷在皮膚傷口癒合過程中,由真皮層的纖維母細胞所表現的Tem1對於皮膚傷口癒合扮演重要的角色。
英文摘要 Tumor endothelial marker 1 (TEM1) is also known as endosialin. It is composed of six domains, a C-type lectin-like domain, a sushi domain, a domain with three epidermal growth factor-like (EGF) repeats, a mucin-like domain, a transmembrane domain, and a cytoplasmic domain. Previous studies demonstrated that Tem1 was expressed abundantly in the skin of mouse embryos, but the expression was quickly down-regulated after birth. However, the functions of Tem1 in skin physiology still need to be further investigated. In vitro study, the scratched wound assay was used on skin fibroblasts to mimic wound healing in vivo. The results revealed that Tem 1 mRNA levels in skin fibroblasts were significantly up-regulated by scratch injury. The similar results were observed when skin fibroblasts were treated with transforming growth factor beta 1 (TGF-β1) through phosphorylating the MAP kinase ERK. Furthermore, cell adhesion, migration, and proliferation were strongly inhibited in Tem1 stable knockdown fibroblasts. In addition, we utilized the Tem1-lacZ knock-in reporter gene mice obtained from Dr. Shu-Wha Lin, National Taiwan University, Taiwan, to screen which cells express Tem1-lacZ encoding β-galactosidase activity in the process of wound healing. The results of in vivo studies showed that β-galactosidase activity was initially detectable in dermis at day 5 and subsequently up-regulated at day 7 and day 9 after skin injury. Results of immunostaining data showed that specific marker of myofibroblasts, α-smooth muscle actin, were detected in β-galactosidase positive cells. Moreover, delayed wound closure was observed in Tem1-knockout mice than wild-type mice. Taken together, our results suggest that TEM-1 in dermal fibroblasts should play a role in the process of cutaneous wound healing.
論文目次 Abstract in Chinese 1
Abstract in English 2
Acknowledgement 3
Contents 4
Figure Contents 6
Appendix Contents 7
Abbreviation 8
Reagents 9
Introduction 12
1. Tumor endothelial marker 1 (TEM1) 12
2. Skin and wound healing process 14
3. The fibroblasts in cutaneous wound healing 16
Specific Aim 17
Material and Method 18
1. Cell culture 18
1-1. Methods of NIH3T3 culture 18
1-2. Methods of M. dunni culture 18
1-3. Cryopreservation of cultured cells 19
1-4. Retrieval of cells from frozen storage 19
2. Western blot assay 19
2-1. Prepare protein sample 19
2-2. Protein quantification 20
2-3. Sodium dodecyl sulfate polyacrylamide gel electrophoresis 20
2-4. Western blot 22
3. RNA extraction 23
4. Reverse transcription-polymerase chain reaction (RT-PCR) 24
5. Polymerase chain reaction (PCR) 25
5-1. Agarose gel electrophoresis 26
6. Tissue genomic DNA extraction 27
7. Tissue staining 28
7-1. X-Gal staining 28
7-2. Paraffin section 29
7-3. Immunohistochemical staining (paraffin) 30
8. Lentiviral infection 31
Results 32
Conclusion 36
Discussion 37
References 40
Figures 45
Appendices 59
Resume 63
參考文獻 Armstrong, D.G., and Jude, E.B. (2002). The role of matrix metalloproteinases in wound healing. Journal of the American Podiatric Medical Association 92, 12-18.
Ashcroft, G.S., Yang, X., Glick, A.B., Weinstein, M., Letterio, J.J., Mizel, D.E., Anzano, M., Greenwell-Wild, T., Wahl, S.M., Deng, C., et al. (1999). Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nat Cell Biol 1, 260-266.
Bainbridge, P. (2013). Wound healing and the role of fibroblasts. Journal of wound care 22, 407-408, 410-412.
Cheng, T.L., Wu, Y.T., Lai, C.H., Kao, Y.C., Kuo, C.H., Liu, S.L., Hsu, Y.Y., Chen, P.K., Cho, C.F., Wang, K.C., et al. (2013). Thrombomodulin regulates keratinocyte differentiation and promotes wound healing. The Journal of investigative dermatology 133, 1638-1645.
Christian, S., Winkler, R., Helfrich, I., Boos, A.M., Besemfelder, E., Schadendorf, D., and Augustin, H.G. (2008). Endosialin (Tem1) is a marker of tumor-associated myofibroblasts and tumor vessel-associated mural cells. The American journal of pathology 172, 486-494.
Conway, E.M., Van de Wouwer, M., Pollefeyt, S., Jurk, K., Van Aken, H., De Vriese, A., Weitz, J.I., Weiler, H., Hellings, P.W., Schaeffer, P., et al. (2002). 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 196, 565-577.
Darby, I., Skalli, O., and Gabbiani, G. (1990). Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing. Laboratory investigation; a journal of technical methods and pathology 63, 21-29.
Darby, I.A., and Hewitson, T.D. (2007). Fibroblast differentiation in wound healing and fibrosis. International review of cytology 257, 143-179.
Derynck, R., and Zhang, Y.E. (2003). Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425, 577-584.
Desmouliere, A., Geinoz, A., Gabbiani, F., and Gabbiani, G. (1993). Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. The Journal of cell biology 122, 103-111.
Dolznig, H., Schweifer, N., Puri, C., Kraut, N., Rettig, W.J., Kerjaschki, D., and Garin-Chesa, P. (2005). Characterization of cancer stroma markers: in silico analysis of an mRNA expression database for fibroblast activation protein and endosialin. Cancer immunity 5, 10.
Dvorak, H.F. (1986). Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. The New England journal of medicine 315, 1650-1659.
Evans, R.A., Tian, Y.C., Steadman, R., and Phillips, A.O. (2003). TGF-beta1-mediated fibroblast-myofibroblast terminal differentiation-the role of Smad proteins. Experimental cell research 282, 90-100.
Falanga, V. (2005). Wound healing and its impairment in the diabetic foot. Lancet 366, 1736-1743.
Fuchs, E. (1995). Keratins and the skin. Annual review of cell and developmental biology 11, 123-153.
Grinnell, F. (1994). Fibroblasts, myofibroblasts, and wound contraction. The Journal of cell biology 124, 401-404.
Haniffa, M.A., Collin, M.P., Buckley, C.D., and Dazzi, F. (2009). Mesenchymal stem cells: the fibroblasts' new clothes? Haematologica 94, 258-263.
Huang, H.P., Hong, C.L., Kao, C.Y., Lin, S.W., Lin, S.R., Wu, H.L., Shi, G.Y., You, L.R., Wu, C.L., and Yu, I.S. (2011). Gene targeting and expression analysis of mouse Tem1/endosialin using a lacZ reporter. Gene expression patterns : GEP 11, 316-326.
Itoh, S., Itoh, F., Goumans, M.J., and Ten Dijke, P. (2000). Signaling of transforming growth factor-beta family members through Smad proteins. European journal of biochemistry / FEBS 267, 6954-6967.
Kondo, T., and Ishida, Y. (2010). Molecular pathology of wound healing. Forensic science international 203, 93-98.
Lane, A., Johnson, D.W., Pat, B., Winterford, C., Endre, Z., Wei, M., and Gobe, G.C. (2002). Interacting roles of myofibroblasts, apoptosis and fibrogenic growth factors in the pathogenesis of renal tubulo-interstitial fibrosis. Growth factors (Chur, Switzerland) 20, 109-119.
MacFadyen, J., Savage, K., Wienke, D., and Isacke, C.M. (2007). Endosialin is expressed on stromal fibroblasts and CNS pericytes in mouse embryos and is downregulated during development. Gene expression patterns : GEP 7, 363-369.
MacFadyen, J.R., Haworth, O., Roberston, D., Hardie, D., Webster, M.T., Morris, H.R., Panico, M., Sutton-Smith, M., Dell, A., van der Geer, P., et al. (2005). Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium. FEBS letters 579, 2569-2575.
Martin, P. (1997). Wound healing--aiming for perfect skin regeneration. Science (New York, NY) 276, 75-81.
Massague, J. (2000). How cells read TGF-beta signals. Nature reviews Molecular cell biology 1, 169-178.
Masuyama, N., Hanafusa, H., Kusakabe, M., Shibuya, H., and Nishida, E. (1999). Identification of two Smad4 proteins in Xenopus. Their common and distinct properties. The Journal of biological chemistry 274, 12163-12170.
Midgley, A.C., Rogers, M., Hallett, M.B., Clayton, A., Bowen, T., Phillips, A.O., and Steadman, R. (2013). Transforming growth factor-beta1 (TGF-beta1)-stimulated fibroblast to myofibroblast differentiation is mediated by hyaluronan (HA)-facilitated epidermal growth factor receptor (EGFR) and CD44 co-localization in lipid rafts. The Journal of biological chemistry 288, 14824-14838.
Moustakas, A., Souchelnytskyi, S., and Heldin, C.H. (2001). Smad regulation in TGF-beta signal transduction. Journal of cell science 114, 4359-4369.
Mustoe, T.A., Pierce, G.F., Thomason, A., Gramates, P., Sporn, M.B., and Deuel, T.F. (1987). Accelerated healing of incisional wounds in rats induced by transforming growth factor-beta. Science (New York, NY) 237, 1333-1336.
Nanda, A., Karim, B., Peng, Z., Liu, G., Qiu, W., Gan, C., Vogelstein, B., St Croix, B., Kinzler, K.W., and Huso, D.L. (2006). Tumor endothelial marker 1 (Tem1) functions in the growth and progression of abdominal tumors. Proceedings of the National Academy of Sciences of the United States of America 103, 3351-3356.
Nepomuceno, R.R., Ruiz, S., Park, M., and Tenner, A.J. (1999). C1qRP is a heavily O-glycosylated cell surface protein involved in the regulation of phagocytic activity. Journal of immunology (Baltimore, Md : 1950) 162, 3583-3589.
Opavsky, R., Haviernik, P., Jurkovicova, D., Garin, M.T., Copeland, N.G., Gilbert, D.J., Jenkins, N.A., Bies, J., Garfield, S., Pastorekova, S., et al. (2001). Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo. The Journal of biological chemistry 276, 38795-38807.
Peterson, J.J., Rayburn, H.B., Lager, D.J., Raife, T.J., Kealey, G.P., Rosenberg, R.D., and Lentz, S.R. (1999). Expression of thrombomodulin and consequences of thrombomodulin deficiency during healing of cutaneous wounds. The American journal of pathology 155, 1569-1575.
Pierce, G.F., Mustoe, T.A., Altrock, B.W., Deuel, T.F., and Thomason, A. (1991). Role of platelet-derived growth factor in wound healing. Journal of cellular biochemistry 45, 319-326.
Ravanti, L., and Kahari, V.M. (2000). Matrix metalloproteinases in wound repair (review). International journal of molecular medicine 6, 391-407.
Rettig, W.J., Garin-Chesa, P., Healey, J.H., Su, S.L., Jaffe, E.A., and Old, L.J. (1992). Identification of endosialin, a cell surface glycoprotein of vascular endothelial cells in human cancer. Proceedings of the National Academy of Sciences of the United States of America 89, 10832-10836.
Robles, D.T., and Berg, D. (2007). Abnormal wound healing: keloids. Clinics in dermatology 25, 26-32.
Rouleau, C., Curiel, M., Weber, W., Smale, R., Kurtzberg, L., Mascarello, J., Berger, C., Wallar, G., Bagley, R., Honma, N., et al. (2008). Endosialin protein expression and therapeutic target potential in human solid tumors: sarcoma versus carcinoma. Clinical cancer research : an official journal of the American Association for Cancer Research 14, 7223-7236.
Rupp, C., Dolznig, H., Puri, C., Sommergruber, W., Kerjaschki, D., Rettig, W.J., and Garin-Chesa, P. (2006). Mouse endosialin, a C-type lectin-like cell surface receptor: expression during embryonic development and induction in experimental cancer neoangiogenesis. Cancer immunity 6, 10.
Ruszczak, Z. (2003). Effect of collagen matrices on dermal wound healing. Advanced drug delivery reviews 55, 1595-1611.
Sadler, J.E. (1997). Thrombomodulin structure and function. Thrombosis and haemostasis 78, 392-395.
Santoro, M.M., and Gaudino, G. (2005). Cellular and molecular facets of keratinocyte reepithelization during wound healing. Experimental cell research 304, 274-286.
Schreier, T., Degen, E., and Baschong, W. (1993). Fibroblast migration and proliferation during in vitro wound healing. A quantitative comparison between various growth factors and a low molecular weight blood dialysate used in the clinic to normalize impaired wound healing. Research in experimental medicine Zeitschrift fur die gesamte experimentelle Medizin einschliesslich experimenteller Chirurgie 193, 195-205.
Sidhanee, A.C. (1983). Structure and function of the skin. Nursing 2, 239-242.
Singer, A.J., and Clark, R.A. (1999). Cutaneous wound healing. The New England journal of medicine 341, 738-746.
Skalli, O., Ropraz, P., Trzeciak, A., Benzonana, G., Gillessen, D., and Gabbiani, G. (1986). A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation. The Journal of cell biology 103, 2787-2796.
St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K.E., Montgomery, E., Lal, A., Riggins, G.J., Lengauer, C., Vogelstein, B., et al. (2000). Genes expressed in human tumor endothelium. Science (New York, NY) 289, 1197-1202.
Suresh Babu, S., Valdez, Y., Xu, A., O'Byrne, A.M., Calvo, F., Lei, V., and Conway, E.M. (2014). TGFbeta-mediated suppression of CD248 in non-cancer cells via canonical Smad-dependent signaling pathways is uncoupled in cancer cells. BMC cancer 14, 113.
Thannickal, V.J., Lee, D.Y., White, E.S., Cui, Z., Larios, J.M., Chacon, R., Horowitz, J.C., Day, R.M., and Thomas, P.E. (2003). Myofibroblast differentiation by transforming growth factor-beta1 is dependent on cell adhesion and integrin signaling via focal adhesion kinase. The Journal of biological chemistry 278, 12384-12389.
Tomkowicz, B., Rybinski, K., Foley, B., Ebel, W., Kline, B., Routhier, E., Sass, P., Nicolaides, N.C., Grasso, L., and Zhou, Y. (2007). Interaction of endosialin/TEM1 with extracellular matrix proteins mediates cell adhesion and migration. Proceedings of the National Academy of Sciences of the United States of America 104, 17965-17970.
Tomkowicz, B., Rybinski, K., Sebeck, D., Sass, P., Nicolaides, N.C., Grasso, L., and Zhou, Y. (2010). Endosialin/TEM-1/CD248 regulates pericyte proliferation through PDGF receptor signaling. Cancer biology & therapy 9, 908-915.
Werner, S., Krieg, T., and Smola, H. (2007). Keratinocyte-fibroblast interactions in wound healing. The Journal of investigative dermatology 127, 998-1008.
Yu, L., Hebert, M.C., and Zhang, Y.E. (2002). TGF-beta receptor-activated p38 MAP kinase mediates Smad-independent TGF-beta responses. The EMBO journal 21, 3749-3759.
Zelensky, A.N., and Gready, J.E. (2005). The C-type lectin-like domain superfamily. The FEBS journal 272, 6179-6217.

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