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

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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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