跳到主要內容

簡易檢索 / 詳目顯示

回結果列表
研究生： 陳曉蓉
Hsiao-Jung Chen
論文名稱： CRSBP-1調節淋巴管內皮細胞移動機制的角色及
Role of CRSBP-1 in Regulation of Cell Migration in Lymphatic Endothelial Cells and its Orthologue in Zebrafish
指導教授： 黃榮三
Jung-San Huang
口試委員:
學位類別： 碩士
Master
系所名稱： 生醫理工學院 - 系統生物與生物資訊研究所
Graduate Institute of Systems Biology and Bioinformatics
畢業學年度： 97
語文別： 英文
論文頁數： 81
中文關鍵詞： 斑馬魚 、淋巴管新生 、淋巴管內皮細胞
外文關鍵詞： zebrafish, lymphatic endothelial cell, lymphangiogenesis, LYVE-1, CRSBP-1
相關次數： 點閱：8  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 我們假設CRSBP-1和它的結合子不只能調節組織間液流動，也參與淋巴管新生。由於淋巴管新生機制包含淋巴管內皮細胞移動、分化，我們便利用細胞移動實驗 (in vitro scratch assay) 測定CRSBP-1專一性結合子調節淋巴管內皮細胞(SVEC4-10 細胞) 之分子機制。我們則證明CRSBP-1專一性結合子 (PDGF peptide和VEGF peptide) ， 促發炎反應細胞激素 (TNF-α)， 及PDGF-BB 會刺激SVEC4-10細胞移動。並且證明PDGF peptide 或 VEGF peptide 是經由與 PDGF-BB不同機制而進而促進淋巴管內皮 (LEC) 細胞移動。除此之外，PDGF peptide在細胞有TNF-α存在下，會保護CRSBP-1不被TNF-α 抑制並且PDGF peptide依然能夠促進細胞移動。最後，我們在斑馬魚當中找出人類CRSBP-1的直系同源蛋白 (orthologue)，是由328個胺基酸組成。衍生斑馬魚CRSBP-1胺基酸序列與人類有26% 相同性，其存在斑馬魚7號染色體上，含有6個外顯子，5個內顯子。斑馬魚CRSBP-1的基因構造和在脊椎動物內的直系同源蛋白相似程度高。我們利用morpholino降低 (knock-down) 該基因在斑馬魚的表現並且得到初步的結果表示CRSBP-1在淋巴管新生分子機制中扮演一個重要角色。

    We hypothesize that CRSBP-1 and its ligands not only regulate interstitial-lymphatic flow but also play a role in lymphangiogenesis. Since lymphangiogenesis involves cell migration and proliferation of LECs, the 1st goal of this thesis work is to determine the role of specific CRSBP-1 ligands in regulation of cell migration in SVEC4-10 cells (lymphatic endothelial cells) by using an in vitro cell migration assay. Here we demonstrate that specific CRSBP-1 ligands (PDGF peptide, VEGF peptide), PDGF-BB and TNF-α stimulate cell migration in SVEC4-10 cells, that specific CRSBP-1 ligands, PDGF-BB and TNF-α stimulate cell migration in these cells through different mechanisms and that PDGF peptide protects cell-surface CRSBP-1 from TNF-α-induced down-regulation and suppresses TNF-α-stimulated cell migration in these cells. The 2nd goal of this thesis is to identify zebrafish CRSBP-1 and its function in zebrafish. Here we demonstrate that the deduced amino acid sequence and genomic structure of zebrafish CRSBP-1 identified are similar to those of other vertebrate orthologues. The preliminary results from experiments using morpholino down-regulation support the hypothesis that CRSBP-1 plays a role in lymphangiogenesis.

    Table of Contents 摘要 .......................................................................................................... i Abstract .................................................................................................. ii Abbreviations ........................................................................................ iii List of Figures ....................................................................................... iv Part 1: Role of CRSBP-1 in regulation of cell migration in SVEC4-10 cells ..................................................................................... 6 1.1 Introduction .................................................................................................... 6 1.1.1 Interstitial-lymphatic flow is important in interstitial fluid homeostasis and cell trafficking ................................................................ 2 1.1.2 The lymphatic system is involved in tumor metastasis ............... 3 1.1.3 CRSBP-1 plays an important role in the function of the lymphatic system ......................................................................................... 5 1.1.4 CRSBP-1 ligands stimulate cell migration in lymphatic endothelial cells (SVEC4-10 cells) ........................................................... 7 1.2 Methods and Materials ................................................................................ 9 1.2.1 Cell culture.......................................................................................... 9 1.2.2 In vitro scratch assay ........................................................................ 9 1.2.3 Immunofluorescent staining of CRSBP-1 .................................... 10 1.2.4 Immunofluorescent staining of F-actin ......................................... 11 1.2.5 Flow cytometry ................................................................................. 12 1.2.6 Statistical analysis ........................................................................... 12 1.3 Results .......................................................................................................... 13 1.3.1 Specific CRSBP-1 ligands stimulate cell migration in SVEC4-10 cells.............................................................................................................. 13 1.3.2 The PDGF β- type receptor (PDGFβR) is involved in CRSBP-1 ligand-stimulated cell migration in SVEC4-10 cells ............................. 18 1.3.3 CRSBP-1 ligands stimulate redistribution of F-actin, which is involved cell migration, in SVEC4-10 cells ............................................ 26 1.3.4 Specific CRSBP-1 ligands confer resistance to TNF-α-stimulated down-regulation of cell-surface CRSBP-1 in SVEC4-10 cells.......................................................................................... 29 1.4 Discussion.................................................................................................... 38 Part 2: Identification and function of the CRSBP-1 orthologue in zebrafish ............................................................................................... 43 2.1 Introduction .................................................................................................. 43 2.2 Methods and Materials .............................................................................. 45 2.2.1 Identification of Zebrafish CRSBP-1 ............................................ 45 2.2.2 Multiple alignment ........................................................................... 45 2.2.3 Phylogenic tree ................................................................................ 45 2.2.4 Kyte-Doolittle hydropathy plot ....................................................... 45 2.2.5 The predicted three-dimentional structure of Link domain in zebrafish CRSBP-1 ................................................................................... 46 2.3 Results and Discussions ........................................................................... 47 2.3.1 Zebrafish CRSBP-1 is a type I membrane glycoprotein containing 4 half-cystine residues. ......................................................... 47 2.3.2 Genomic structure of Zebrafish CRSBP-1 .................................. 56 2.3.3 Function of Zebrafish CRSBP-1 ................................................... 59 Part 3: Conclusions ............................................................................. 66 References ........................................................................................... 68

    References
    1. Grundy, S.M. and H.Y. Mok, Chylomicron clearance in normal and hyperlipidemic man. Metabolism, 1976. 25(11): p. 1225-39.
    2. Schmid-Schonbein, G.W., Microlymphatics and lymph flow. Physiol Rev, 1990. 70(4): p. 987-1028.
    3. Cao, R., et al., PDGF-BB induces intratumoral lymphangiogenesis and promotes lymphatic metastasis. Cancer Cell, 2004. 6(4): p. 333-45.
    4. Leak, L.V., The structure of lymphatic capillaries in lymph formation. Fed Proc, 1976. 35(8): p. 1863-71.
    5. Makinen, T., C. Norrmen, and T.V. Petrova, Molecular mechanisms of lymphatic vascular development. Cell Mol Life Sci, 2007. 64(15): p. 1915-29.
    6. Gerli, R., et al., Specific adhesion molecules bind anchoring filaments and endothelial cells in human skin initial lymphatics. Lymphology, 2000. 33(4): p. 148-57.
    7. Baluk, P., et al., Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med, 2007. 204(10): p. 2349-62.
    8. Alitalo, K. and P. Carmeliet, Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell, 2002. 1(3): p. 219-27.
    9. Tobler, N.E. and M. Detmar, Tumor and lymph node lymphangiogenesis--impact on cancer metastasis. J Leukoc Biol, 2006. 80(4): p. 691-6.
    10. Dyer, M.A., et al., Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina. Nat Genet, 2003. 34(1): p. 53-8.
    11. Sleeman, J., A. Schmid, and W. Thiele, Tumor lymphatics. Semin Cancer Biol,
    69
    2009.
    12. Heldin, C.H., et al., High interstitial fluid pressure - an obstacle in cancer therapy. Nat Rev Cancer, 2004. 4(10): p. 806-13.
    13. Salnikov, A.V., et al., Lowering of tumor interstitial fluid pressure specifically augments efficacy of chemotherapy. FASEB J, 2003. 17(12): p. 1756-8.
    14. Karpanen, T., et al., Vascular endothelial growth factor C promotes tumor lymphangiogenesis and intralymphatic tumor growth. Cancer Res, 2001. 61(5): p. 1786-90.
    15. Huang, S.S., et al., Cloning, expression, characterization, and role in autocrine cell growth of cell surface retention sequence binding protein-1. J Biol Chem, 2003. 278(44): p. 43855-69.
    16. Boensch, C., et al., Identification, purification, and characterization of cell-surface retention sequence-binding proteins from human SK-Hep cells and bovine liver plasma membranes. J Biol Chem, 1995. 270(4): p. 1807-16.
    17. Banerji, S., et al., LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol, 1999. 144(4): p. 789-801.
    18. Jackson, D.G., Biology of the lymphatic marker LYVE-1 and applications in research into lymphatic trafficking and lymphangiogenesis. APMIS, 2004. 112(7-8): p. 526-38.
    19. Huang, S.S., et al., CRSBP-1/LYVE-l-null mice exhibit identifiable morphological and functional alterations of lymphatic capillary vessels. FEBS Lett, 2006. 580(26): p. 6259-68.
    20. Rivera, G.M., et al., Requirement of Nck adaptors for actin dynamics and cell migration stimulated by platelet-derived growth factor B. Proc Natl Acad Sci U S A, 2006. 103(25): p. 9536-41.
    21. Ruusala, A., et al., Nck adapters are involved in the formation of dorsal ruffles, cell migration, and Rho signaling downstream of the platelet-derived growth factor beta receptor. J Biol Chem, 2008. 283(44): p. 30034-44.
    70
    22. Abouantoun, T.J. and T.J. Macdonald, Imatinib blocks migration and invasion of medulloblastoma cells by concurrently inhibiting activation of platelet-derived growth factor receptor and transactivation of epidermal growth factor receptor. Mol Cancer Ther, 2009.
    23. Cao, Y., Direct role of PDGF-BB in lymphangiogenesis and lymphatic metastasis. Cell Cycle, 2005. 4(2): p. 228-30.
    24. Johnson, L.A., et al., Inflammation-induced uptake and degradation of the lymphatic endothelial hyaluronan receptor LYVE-1. J Biol Chem, 2007. 282(46): p. 33671-80.
    25. Jiang, W., P. Desjardins, and R.F. Butterworth, Cerebral inflammation contributes to encephalopathy and brain edema in acute liver failure: protective effect of minocycline. J Neurochem, 2009. 109(2): p. 485-93.
    26. Wolf, N.B., et al., Influences of opioids and nanoparticles on in vitro wound healing models, in Eur J Pharm Biopharm. 2009.
    27. Adams, R.H. and K. Alitalo, Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol, 2007. 8(6): p. 464-78.
    28. Cleaver, O. and D.A. Melton, Endothelial signaling during development. Nat Med, 2003. 9(6): p. 661-8.
    29. Ny, A., et al., A genetic Xenopus laevis tadpole model to study lymphangiogenesis. Nat Med, 2005. 11(9): p. 998-1004.
    30. Garrafa, E., et al., Isolation and characterization of lymphatic microvascular endothelial cells from human tonsils. J Cell Physiol, 2006. 207(1): p. 107-13.
    31. Kuchler, A.M., et al., Development of the zebrafish lymphatic system requires VEGFC signaling. Curr Biol, 2006. 16(12): p. 1244-8.
    32. Yaniv, K., et al., Live imaging of lymphatic development in the zebrafish. Nat Med, 2006. 12(6): p. 711-6.
    33. Isogai, S., et al., Zebrafish as a new animal model to study lymphangiogenesis. Anat Sci Int, 2009.
    71
    34. McKinney, M.C. and B.M. Weinstein, Chapter 4. Using the zebrafish to study vessel formation. Methods Enzymol, 2008. 444: p. 65-97.
    35. Nightingale, T.D., et al., A mechanism of sialylation functionally silences the hyaluronan receptor LYVE-1 in lymphatic endothelium. J Biol Chem, 2009. 284(6): p. 3935-45.
    36 Wei-Hsien Hou, phD Dissertation, Saint Louis University School of Medicine, 2009

    QR CODE
    :::