CRSBP-1調節淋巴管內皮細胞移動機制的角色及
其斑馬魚直系同源蛋白

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陳曉蓉(Hsiao-Jung Chen) 查詢紙本館藏

畢業系所

系統生物與生物資訊研究所

論文名稱

CRSBP-1調節淋巴管內皮細胞移動機制的角色及其斑馬魚直系同源蛋白
(Role of CRSBP-1 in Regulation of Cell Migration in Lymphatic Endothelial Cells and its Orthologue in Zebrafish )

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★ 以CRSBP-1接合子調控巨噬細胞的移動及吞噬

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 斑馬魚
★ 淋巴管新生
★ 淋巴管內皮細胞

關鍵字(英)

★ zebrafish
★ lymphatic endothelial cell
★ lymphangiogenesis
★ LYVE-1
★ CRSBP-1

論文目次

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

參考文獻

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

指導教授

黃榮三(Jung-San Huang)

審核日期

2009-7-23

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