進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-0807201314005900
論文名稱(中文) 探討凝血酶調節素在眼角膜上皮細胞傷口癒合的角色
論文名稱(英文) The Role of Thrombomodulin in Corneal Epithelial 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 101
學期 2
出版年 102
研究生(中文) 翼景城
研究生(英文) Ching-Chang I
電子信箱 caesar0312@gmail.com
學號 s16001036
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-蔡曜聲
中文關鍵字 凝血酶調節素  眼角膜上皮細胞  傷口癒合 
英文關鍵字 Thrombomodulin  corneal epithelium  wound healing 
學科別分類
中文摘要 凝血酶調節素(thrombomodulin,TM)為一穿膜的醣蛋白,在表皮細胞以及內皮細胞中高度表達。TM具有調控凝血、發炎、表皮細胞的分化和細胞生長的功能。角膜,為一透明、沒有血管的組織,位於眼球的前半部,其功能為讓光產生屈折和提供屏障使異物無法進入眼球內部。角膜大致上可以分為三層:最外層的表皮細胞層(epithelium)、中間的基質層(stroma)以及最底層單層的內皮細胞層(endothelium)。之前的研究指出,疾病狀態如眼內炎(endophthalmitis)、病毒性角膜炎(herpetic keratitis)的人類眼球角膜組織中會有TM的表現,這顯示TM可能與角膜的發炎有關。因此,我們假設TM在角膜中可能扮演調控發炎以及組織再生的角色。然而,TM在角膜表現分布以及確切功能尚未清楚。在本實驗中,藉由免疫螢光染色觀察TM在小鼠角膜上皮細胞的表現分布,並利用角膜刮除(corneal debridement )以及鹼性化學燒傷 (alkali burn) 動物模式研究TM在角膜組織修復及發炎的功能。結果顯示,小鼠的角膜上皮細胞層及內皮細胞層均有TM的表現。此外,也觀察到TM在角膜上皮的傷口癒合過程中表現量上升。細胞實驗中,使用小鼠的初代培養角膜上皮細胞加入血小板衍生性生長因子 (PDGF-BB) 後發現TM的表現量上升。另外,我們使用重組蛋白rTMD23 (recombinant TM domain 23) 在實驗中發現可以促進角膜傷口的癒合。總括而言,我們首次建立了TM在正常小鼠角膜上皮細胞以及傷口癒合過程的表現特徵。更進一步推論,TM在角膜上皮細胞傷口癒合過程中具有功能且可能是藉由PDGF-BB的調控。
英文摘要 Thrombomodulin (TM) is a transmembrane glycoprotein highly expressed in endothelial cells and epithelial cells. TM participates in regulation of coagulation, inflammation, epithelial differentiation and cell proliferation. Cornea, a transparent tissue in the front part of the eye, provides barrier function and refraction of light. Previous study showed that TM is expressed in corneal epithelium of diseases associated with endophthalmitis and herpetic keratitis, suggesting that TM may regulate corneal inflammation. However, the distribution and the regulation of TM expression in cornea remain largely uncertain. Therefore, we hypothesize that TM is expressed in corneal epithelium where it regulates corneal regeneration and inflammation. In this study, the expression of TM in mouse cornea was examined by using immunofluorescent staining and the effect of TM in corneal regeneration and inflammation were evaluated by mouse model of corneal epithelial debridement and alkali-induced corneal injury. Our result showed that TM was expressed in the corneal epithelium and corneal endothelium in normal adult mice. In addition, the expression of TM was increased in the early phase of wound healing and it was decreased after wound recovery. In the ex vivo study, we found that platelet-derived growth factor-BB (PDGF-BB) induced TM expression in primary mouse corneal epithelial cells (MCECs). In addition, the administration of recombinant TM epidermal growth factor-like domain plus serine/threonine-rich domain (rTMD23) increased corneal wound healing in mouse model of corneal debridement wound. In conclusion, the expression of TM in corneal epithelium was modulated during corneal wound healing and that may be regulated by PDGF-BB. Additionally, the recombinant TM epidermal growth factor-like domain plus serine/threonine-rich domain promoted corneal wound healing.
論文目次
Acknowledgement 1
Abstract in Chinese 2
Abstract in English 3
Content 4
Figure Contents 5
Abbreviation 6
Instruments 7
Reagents and Chemicals 9
Introduction 13
Specific Aim 18
Materials and Methods 19
Results 34
Conclusion 38
Discussion 39
Figures 44
Appendixes 53
Author’s resume 56
References 57
參考文獻 1. Abeyama, K., Stern, D.M., Ito, Y., Kawahara, K., Yoshimoto, Y., Tanaka, M., Uchimura, T., Ida, N., Yamazaki, Y., Yamada, S., et al. (2005). The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. The Journal of clinical investigation 115, 1267-1274.
2. Agrawal, V.B., and Tsai, R.J. (2003). Corneal epithelial wound healing. Indian J Ophthalmol 51, 5-15.
3. Buck, R.C. (1979). Cell migration in repair of mouse corneal epithelium. Investigative ophthalmology & visual science 18, 767-784.
4. Chee, K.Y., Kicic, A., and Wiffen, S.J. (2006). Limbal stem cells: the search for a marker. Clinical & experimental ophthalmology 34, 64-73.
5. 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.
6. Cheng, T.L., Wu, Y.T., Lin, H.Y., Hsu, F.C., Liu, S.K., Chang, B.I., Chen, W.S., Lai, C.H., Shi, G.Y., and Wu, H.L. (2011). Functions of rhomboid family protease RHBDL2 and thrombomodulin in wound healing. The Journal of investigative dermatology 131, 2486-2494.
7. Chung, E.H., Hutcheon, A.E., Joyce, N.C., and Zieske, J.D. (1999). Synchronization of the G1/S transition in response to corneal debridement. Investigative ophthalmology & visual science 40, 1952-1958.
8. Chusid, M.J., and Davis, S.D. (1979). Experimental bacterial keratitis in neutropenic guinea pigs: polymorphonuclear leukocytes in corneal host defense. Infection and immunity 24, 948-952.
9. Conway, E.M. (2012). Thrombomodulin and its role in inflammation. Seminars in immunopathology 34, 107-125.
10. Conway, E.M., Nowakowski, B., and Steiner-Mosonyi, M. (1992). Human neutrophils synthesize thrombomodulin that does not promote thrombin-dependent protein C activation. Blood 80, 1254-1263.
11. 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.
12. Daimon, T., Kazama, M., Miyajima, Y., and Nakano, M. (1997). Immunocytochemical localization of thrombomodulin in the aqueous humor passage of the rat eye. Histochemistry and cell biology 108, 121-131.
13. Esmon, C.T. (1995). Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 9, 946-955.
14. Haber, M., Cao, Z., Panjwani, N., Bedenice, D., Li, W.W., and Provost, P.J. (2003). Effects of growth factors (EGF, PDGF-BB and TGF-beta 1) on cultured equine epithelial cells and keratocytes: implications for wound healing. Veterinary ophthalmology 6, 211-217.
15. Hamada, H., Ishii, H., Sakyo, K., Horie, S., Nishiki, K., and Kazama, M. (1995). The epidermal growth factor-like domain of recombinant human thrombomodulin exhibits mitogenic activity for Swiss 3T3 cells. Blood 86, 225-233.
16. Healy, A.M., Rayburn, H.B., Rosenberg, R.D., and Weiler, H. (1995). 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 92, 850-854.
17. Hoppenreijs, V.P., Pels, E., Vrensen, G.F., Felten, P.C., and Treffers, W.F. (1993). Platelet-derived growth factor: receptor expression in corneas and effects on corneal cells. Investigative ophthalmology & visual science 34, 637-649.
18. Hsu, Y.Y., Shi, G.Y., Kuo, C.H., Liu, S.L., Wu, C.M., Ma, C.Y., Lin, F.Y., Yang, H.Y., and Wu, H.L. (2012). Thrombomodulin is an ezrin-interacting protein that controls epithelial morphology and promotes collective cell migration. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 26, 3440-3452.
19. Huang, H.C., Shi, G.Y., Jiang, S.J., Shi, C.S., Wu, C.M., Yang, H.Y., and Wu, H.L. (2003). Thrombomodulin-mediated cell adhesion: involvement of its lectin-like domain. The Journal of biological chemistry 278, 46750-46759.
20. Ikeda, T., Ishii, H., Higuchi, T., Sato, K., Hayashi, Y., Ikeda, K., and Hirabayashi, Y. (2000). Localization of thrombomodulin in the anterior segment of the human eye. Investigative ophthalmology & visual science 41, 3383-3390.
21. Imanishi, J., Kamiyama, K., Iguchi, I., Kita, M., Sotozono, C., and Kinoshita, S. (2000). Growth factors: importance in wound healing and maintenance of transparency of the cornea. Progress in retinal and eye research 19, 113-129.
22. Ito, T., Kawahara, K., Okamoto, K., Yamada, S., Yasuda, M., Imaizumi, H., Nawa, Y., Meng, X., Shrestha, B., Hashiguchi, T., et al. (2008). Proteolytic cleavage of high mobility group box 1 protein by thrombin-thrombomodulin complexes. Arteriosclerosis, thrombosis, and vascular biology 28, 1825-1830.
23. Kamiyama, K., Iguchi, I., Wang, X., and Imanishi, J. (1998). Effects of PDGF on the migration of rabbit corneal fibroblasts and epithelial cells. Cornea 17, 315-325.
24. Kawamura, H., Hiramatsu, Y., and Watanabe, I. (1996). Localization of thrombomodulin in a rabbit eye. Curr Eye Res 15, 938-942.
25. Keino, H., Yamakawa, N., Goto, H., Usui, M., and Nakano, M. (1998). [Measurement of thrombomodulin values in the serum and eyes of experimental autoimmune uveoretinitis]. Nippon Ganka Gakkai zasshi 102, 28-33.
26. Klenkler, B., and Sheardown, H. (2004). Growth factors in the anterior segment: role in tissue maintenance, wound healing and ocular pathology. Experimental eye research 79, 677-688.
27. Klenkler, B., Sheardown, H., and Jones, L. (2007). Growth factors in the tear film: role in tissue maintenance, wound healing, and ocular pathology. The ocular surface 5, 228-239.
28. Klyce, S.D. (1972). Electrical profiles in the corneal epithelium. The Journal of physiology 226, 407-429.
29. Kobayashi, T., Yoshioka, R., Shiraishi, A., and Ohashi, Y. (2009). New technique for culturing corneal epithelial cells of normal mice. Molecular vision 15, 1589-1593.
30. Lager, D.J., Callaghan, E.J., Worth, S.F., Raife, T.J., and Lentz, S.R. (1995). Cellular localization of thrombomodulin in human epithelium and squamous malignancies. Am J Pathol 146, 933-943.
31. Li, Y.H., Kuo, C.H., Shi, G.Y., and Wu, H.L. (2012). The role of thrombomodulin lectin-like domain in inflammation. Journal of biomedical science 19, 34.
32. Li, Z., Burns, A.R., and Smith, C.W. (2006). Two waves of neutrophil emigration in response to corneal epithelial abrasion: distinct adhesion molecule requirements. Investigative ophthalmology & visual science 47, 1947-1955.
33. Liang, C.C., You, L.R., Chang, J.L., Tsai, T.F., and Chen, C.M. (2009). Transgenic mice exhibiting inducible and spontaneous Cre activities driven by a bovine keratin 5 promoter that can be used for the conditional analysis of basal epithelial cells in multiple organs. Journal of biomedical science 16, 2.
34. Lo, I.C., Lin, T.M., Chou, L.H., Liu, S.L., Wu, L.W., Shi, G.Y., Wu, H.L., and Jiang, M.J. (2009). Ets-1 mediates platelet-derived growth factor-BB-induced thrombomodulin expression in human vascular smooth muscle cells. Cardiovascular research 81, 771-779.
35. Lu, B., Wang, H., Andersson, U., and Tracey, K.J. (2013). Regulation of HMGB1 release by inflammasomes. Protein & cell 4, 163-167.
36. Lyngholm, M., Vorum, H., Nielsen, K., Ostergaard, M., Honore, B., and Ehlers, N. (2008). Differences in the protein expression in limbal versus central human corneal epithelium--a search for stem cell markers. Experimental eye research 87, 96-105.
37. Ma, X., Shimmura, S., Miyashita, H., Yoshida, S., Kubota, M., Kawakita, T., and Tsubota, K. (2009). Long-term culture and growth kinetics of murine corneal epithelial cells expanded from single corneas. Investigative ophthalmology & visual science 50, 2716-2721.
38. Maruyama, I., Bell, C.E., and Majerus, P.W. (1985). Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. The Journal of cell biology 101, 363-371.
39. McCachren, S.S., Diggs, J., Weinberg, J.B., and Dittman, W.A. (1991). Thrombomodulin expression by human blood monocytes and by human synovial tissue lining macrophages. Blood 78, 3128-3132.
40. Meek, K.M., Dennis, S., and Khan, S. (2003). Changes in the refractive index of the stroma and its extrafibrillar matrix when the cornea swells. Biophysical journal 85, 2205-2212.
41. Mizutani, H., Hayashi, T., Nouchi, N., Ohyanagi, S., Hashimoto, K., Shimizu, M., and Suzuki, K. (1994). Functional and immunoreactive thrombomodulin expressed by keratinocytes. The Journal of investigative dermatology 103, 825-828.
42. Mizutani, H., Ohyanagi, S., Hayashi, T., Groves, R.W., Suzuki, K., and Shimizu, M. (1996a). Functional thrombomodulin expression on epithelial skin tumours as a differentiation marker for suprabasal keratinocytes. In The British journal of dermatology, pp. 187-193.
43. Mizutani, H., Ohyanagi, S., Nouchi, N., Inachi, S., and Shimizu, M. (1996b). Tissue factor and thrombomodulin expression on keratinocytes as coagulation/anti-coagulation cofactor and differentiation marker. The Australasian journal of dermatology 37 Suppl 1, S48-49.
44. Muzumdar, M.D., Tasic, B., Miyamichi, K., Li, L., and Luo, L. (2007). A global double-fluorescent Cre reporter mouse. Genesis 45, 593-605.
45. Namba, H., Kashiwagi, Y., Nishitsuka, K., Takamura, H., Yamamoto, T., and Yamashita, H. (2010). Association of PDGF-BB-induced thrombomodulin with the regulation of inflammation in the corneal and scleral stroma. Investigative ophthalmology & visual science 51, 5460-5469.
46. Pajoohesh-Ganji, A., and Stepp, M.A. (2005). In search of markers for the stem cells of the corneal epithelium. Biology of the cell / under the auspices of the European Cell Biology Organization 97, 265-276.
47. 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. Am J Pathol 155, 1569-1575.
48. Raife, T.J., Lager, D.J., Madison, K.C., Piette, W.W., Howard, E.J., Sturm, M.T., Chen, Y., and Lentz, S.R. (1994). Thrombomodulin expression by human keratinocytes. Induction of cofactor activity during epidermal differentiation. The Journal of clinical investigation 93, 1846-1851.
49. Schlotzer-Schrehardt, U., and Kruse, F.E. (2005). Identification and characterization of limbal stem cells. Experimental eye research 81, 247-264.
50. Secker, G.A., and Daniels, J.T. (2008a). Corneal epithelial stem cells: deficiency and regulation. Stem cell reviews 4, 159-168.
51. Secker, G.A., and Daniels, J.T. (2008b). Limbal epithelial stem cells of the cornea. In StemBook (Cambridge (MA)).
52. Shi, C.S., Shi, G.Y., Chang, Y.S., Han, H.S., Kuo, C.H., Liu, C., Huang, H.C., Chang, Y.J., Chen, P.S., and Wu, H.L. (2005). Evidence of human thrombomodulin domain as a novel angiogenic factor. Circulation 111, 1627-1636.
53. Sun, T.T., and Lavker, R.M. (2004). Corneal epithelial stem cells: past, present, and future. The journal of investigative dermatology. Symposium proceedings / the Society for Investigative Dermatology, Inc. [and] European Society for Dermatological Research 9, 202-207.
54. Tracy, P.B. (1988). Regulation of thrombin generation at cell surfaces. Seminars in thrombosis and hemostasis 14, 227-233.
55. Tseng, S.C. (1989). Concept and application of limbal stem cells. Eye 3 ( Pt 2), 141-157.
56. Vande Walle, L., Kanneganti, T.D., and Lamkanfi, M. (2011). HMGB1 release by inflammasomes. Virulence 2, 162-165.
57. Vesaluoma, M., Teppo, A.M., Gronhagen-Riska, C., and Tervo, T. (1997). Platelet-derived growth factor-BB (PDGF-BB) in tear fluid: a potential modulator of corneal wound healing following photorefractive keratectomy. Curr Eye Res 16, 825-831.
58. Wagoner, M.D., Kenyon, K.R., Gipson, I.K., Hanninen, L.A., and Seng, W.L. (1984). Polymorphonuclear neutrophils delay corneal epithelial wound healing in vitro. Investigative ophthalmology & visual science 25, 1217-1220.
59. Wilson, S.E., Mohan, R.R., Mohan, R.R., Ambrosio, R., Jr., Hong, J., and Lee, J. (2001). The corneal wound healing response: cytokine-mediated interaction of the epithelium, stroma, and inflammatory cells. Progress in retinal and eye research 20, 625-637.
60. Yoshioka, R., Shiraishi, A., Kobayashi, T., Morita, S., Hayashi, Y., Higashiyama, S., and Ohashi, Y. (2010). Corneal epithelial wound healing impaired in keratinocyte-specific HB-EGF-deficient mice in vivo and in vitro. Investigative ophthalmology & visual science 51, 5630-5639.
61. Yu, F.S., Yin, J., Xu, K., and Huang, J. (2010). Growth factors and corneal epithelial wound healing. Brain research bulletin 81, 229-235.
62. Zagon, I.S., Sassani, J.W., and McLaughlin, P.J. (1999). Cellular dynamics of corneal wound re-epithelialization in the rat. I. Fate of ocular surface epithelial cells synthesizing DNA prior to wounding. Brain research 822, 149-163.
63. Zhang, J., Huang, C., Feng, Y., Li, Y., and Wang, W. (2012a). Comparison of beneficial factors for corneal wound-healing of rat mesenchymal stem cells and corneal limbal stem cells on the xenogeneic acellular corneal matrix in vitro. Molecular vision 18, 161-173.
64. Zhang, Y., Kobayashi, T., Hayashi, Y., Yoshioka, R., Shiraishi, A., Shirasawa, S., Higashiyama, S., and Ohashi, Y. (2012b). Important role of epiregulin in inflammatory responses during corneal epithelial wound healing. Investigative ophthalmology & visual science 53, 2414-2423.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2023-03-12起公開。
  • 同意授權校外瀏覽/列印電子全文服務,於2023-03-12起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw