以CRSBP-1接合子調控巨噬細胞的移動及吞噬

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姓名

蔡承遠(Cheng-yuan Tsai) 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

以CRSBP-1接合子調控巨噬細胞的移動及吞噬
(Regulation of Macrophage Migration and Phagocytosisby CRSBP-1 Ligands)

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摘要(中)

CRSBP-1 是一種存在於細胞膜的受器。當生長因子（如：血小板生長因子-BB 型及血管生長因子-A 型）由培養的細胞株合成並分泌時，可被此蛋白調控固定於細胞表面。目前並不清楚CRSBP-1 的生理功能。在我們之前的研究，發現同時剔除CRSBP-1 與Apo E 基因的老鼠比只剔除Apo E 基因的老鼠，罹患動脈血管硬化疾病的機率為低，這個發現使我們懷疑CRSBP-1 在動脈血管硬化疾病上或許扮演重要的角色。已知巨噬細胞參與動脈血管硬化疾病的病理過程，且最近被發現巨噬細胞可以表現CRSBP-1，於是我們假設CRSBP-1 和其接合子在動脈血管硬化疾病形成過程中，調控巨噬細胞移動及吞噬功能。在此實驗，我們利用CRSBP-1 的接合子，包括血小板生長因子-BB 型及另外兩個特別的接合子（血小板生長因子-胜肽及血管生長因子-胜肽，專一地結合CRSBP-1，與血小板生長因子-BB 型及血管生長因子-A 型的接受器沒有交互作用），刺激RAW 264.7 類似巨噬細胞株及骨髓衍生巨噬細胞的移動及吞噬能力，然後分別以傷口癒合試驗、Boyden chamber 試驗及吞噬FITC-葡聚醣試驗測定，得出分別在5 和40 ng/mL、10 和20 uM 及10 μg/mL為最適合濃度，且血管生長因子-胜肽出比血小板生長因子-BB 型及血小板生長因子-胜肽有更好的效果，而這種效果可以被酪氨酸激酶抑制劑所抑制。結果表示CRSBP-1 接合子可以刺激巨噬細胞移動及吞噬能力，這或許在動脈血管硬化疾病扮演關鍵的角色。而CRSBP-1 的拮抗劑和血小板生長因子β 型激酶抑制劑，則可以治療和預防動脈血管硬化疾病。

摘要(英)

CRSBP-1 is a membrane receptor which mediates the phenomenon of cell-surface retention of growth factors (e.g., PDGF-BB and VEGF-A) after synthesis and secretion in cultured cells. The physiological function of CRSBP-1 is unknown. In our recent studies, we found that CRSBP-1-/Apo E- mice have lower risk of atherosclerosis disease than CRSBP-1+/Apo E- mice. This finding suggests that CRSBP-1 may play an important role in the pathogenesis of atherosclerosis. Since macrophages are known to be involved in the pathogenesis of atherosclerosis and since CRSBP-1 has recently been found to be expressed in macrophages, we hypothesize that CRSBP-1 and its ligands regulate migration and phagocytosis of macrophages in the process of the atherosclerosis disease. Here we demonstrate that the ligands of CRSBP-1 such as PDGF-BB and two specific ligands (PDGF-peptide and VEGF-peptide which specifically bind to CRSBP-1 and do not interact with the respective growth factor receptors of PDGF-BB and VEGF-A) stimulate migration and phagocytosis in RAW 264.7 cells, a macrophage-like cell line, and bone marrow–derived macrophages (BMM) with the optimal concentrations of 5-40 ng/mL, 10-20 uM and 10 μg/mL, respectively . The migration and phagocytosis are determined by scratch and Boyden chamber assays, and by measuring FITC-dextran uptake, respectively. VEGF-peptide appears to be more potent than PDGF-BB and PDGF-peptide in stimulating macrophage migration and phagocytosis at their optimal concentrations. The effects of these CRSB-1 ligands can be abolished in the presence of a PDGF β-type receptor kianse inhibitor. These results suggest that CRSBP-1 ligand-stimulated migration and phagocytosis of macrophages may play a pivotal role in the pathogenesis of atherosclerosis. These results also suggest that CRSBP-1antasgonists and PDGF β-type kinase inhibitors have potential to be therapeutic agents for treating and preventing atherosclerosis.

關鍵字(中)

★ 吞噬
★ 巨噬細胞
★ 移動

關鍵字(英)

★ Phogocytic uptake
★ Boyden chamber
★ Phagocytosis
★ Migration
★ Macrophage
★ RAW 264.7
★ bone marrow–derived macrophage
★ LYVE-1
★ CRSBP-1
★ Scratch

論文目次

中文摘要 …………………………………………………………… i
Abstract ……………………………………………………………… ii
致謝 ………………………………………………………………… iii
Table of Contents …………………………………………………… iv
List of Figures ………………………………………………………… vi
Abbreviations ………………………………………………………… ix
1 Introduction ……………………………………………………… 1
1.1 Macrophage ………………………………………………… 1
1.1.1 Macrophage activation ……………………………… 2
1.1.1.1 Classically activated macrophages …………… 2
1.1.1.2 Alternatively activated macrophages ………… 3
1.1.2 Macrophage migration ……………………………… 4
1.1.3 Macrophage phagocytosis …………………………… 5
1.1.4 Macrophages are pivotal in the pathogenesis of
atherosclerosis …………………………………………5
1.2 Cell-surface retention sequences binding protein-1
(CRSBP-1) …………………………………………………6
1.2.1 PDGF and VEGF-peptides can be used to study the
biological function of CRSBP-1 ……………………6
1.2.2 Macrophage express CRSBP-1(LYVE-1) …………… 7
1.3 Specific aim ………………………………………………… 8
2 Materials and Methods …………………………………………… 9
2.1 Growth factors, peptides, chemicals and antibodies ………… 9
2.2 Cell-line culture …………………………………………… 9
2.3 Preparation of L929 conditioned medium ………………… 10
2.4 Mouse BMM isolation and culture ………………………… 10
2.5 Scratch assay ………………………………………………… 11
2.6 Boyden chamber assay ……………………………………… 11
2.7 Direct immunofluorescence staining for Fluorescence
Microscopy ………………………………………………12
2.8 Direct Immunofluorescence Staining for Flow Cytometry … 13
2.9 Flow cytometry assay ……………………………………… 13
2.10 Phogocytic uptake assay …………………………………… 13
2.11 Statistics …………………………………………………… 14
3 Results …………………………………………………………… 14
3.1 TNF-α, PDGF-BB, PDGF- peptide and VEGF-peptide
stimulate migration of RAW 264.7 cells …………………14
3.2 TNF-α, PDGF-BB, PDGF- peptide and VEGF-peptide
stimulate migration of bone marrow–derived macrophages
(BMM) ………………………………………………16
3.3 TNF-α, PDGF-BB, PDGF- peptide and VEGF-peptide
stimulate phagocytic uptake of FITC-dextran by RAW 264.7
cells ……………………………………………………17
4 Discussion ………………………………………………………… 17
Figures ………………………………………………………… 20
References …………………………………………………………… 50

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指導教授

凌慶東、黃榮三
(Qing-dong Ling、Jung-san Huang)

審核日期

2010-7-20

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