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系統識別號 U0026-2308201221424000
論文名稱(中文) 多價結構的金奈米粒子-半乳糖凝集素-1複合物 調節細胞凋亡訊息與膠原蛋白誘發關節炎治療效果
論文名稱(英文) Multivalent structure of nanogold-galectin-1 complex regulates apoptosis signaling and therapeutic effects in collagen-induced arthritis
校院名稱 成功大學
系所名稱(中) 基礎醫學研究所
系所名稱(英) Institute of Basic Medical Sciences
學年度 100
學期 2
出版年 101
研究生(中文) 黃彥彰
研究生(英文) Yen-Jang Huang
學號 s58961078
學位類別 博士
語文別 英文
論文頁數 55頁
口試委員 指導教授-吳昭良
口試委員-謝奇璋
口試委員-王崇任
口試委員-戴明泓
口試委員-林崇智
中文關鍵字 類風濕性關節炎  細胞凋亡  galectin-1  金奈米粒子  多價性結構 
英文關鍵字 rheumatoid arthritis  apoptosis  galectin-1  gold nanoparticle  multivalent structure 
學科別分類
中文摘要 細胞的行為是由許多的步驟所控制,包含由配體結合至細胞表面受體所引起的胞內的訊息傳導。多價配體擁有多個可以與受體結合的辨識區,且可以影響下游的訊息。現在,金奈米粒子被高度應用在化學、生物、工程與醫藥。金奈米粒子-配體複合物可以形成多價的結構,藉由提高結合力與專一性去交叉連結受體。Galectin-1對β-galactosides有很高的結合力,且聚集細胞膜上的receptor去傳遞各種胞內訊息。其中,Galectin-1可以誘導特定種類的胸腺細胞與活化的T細胞凋亡。在此,我們建立了擁有多價性結構的金奈米立子-galectin-1複合物 (Au-Gal1),並且期待這複合物多價的特性可以增加比galectin-1或金奈米在類風濕性關節炎的治療上更好的療效。在Galectin-1結合至金奈米後,提高了對Jurkat細胞的CD45聚集且抑制它的去磷酸酶的能力,造成藉由caspase路徑促進細胞凋亡。在膠原蛋白誘發之大鼠關節炎模式中,Au-Gal1被注射入關節內,去促進關節內CD4+ T細胞的凋亡和降低前發炎細胞素的程度,改善關節炎的臨床症狀。這些治療效果顯示,奈米粒子-受體的多價結構可以調節細胞表面受體的分佈與胞內訊息。這對於奈米粒子的應用提供一個嶄新的觀點。
英文摘要 Cellular behaviour is controlled by numerous processes, including intracellular signaling pathways that are triggered by the binding of ligands with cell surface receptors. Multivalent ligands have multiple copies of a recognition element that binds to receptors and influences downstream signals. Gold nanoparticles are now highly utilized in chemistry, biology, engineering, and medicine. Gold nanoparticle-ligand complexes form multivalent structures to crosslink receptors with high avidity and specificity. Galectin-1 has high affinity for β-galactosides and engages with cell surface receptor to deliver a variety of intracellular signals. Galectin-1 induces apoptosis of specific thymocyte subsets and activates T cells. Here, we developed nanogold-galectin-1 (Au-Gal1) complexes with multivalent structure and expect the multivalent property of Au-Gal1 complexes will have more therapeutic potential than free-form galectin-1 or nanogold for rheumatoid arthritis. After conjugation onto gold nanoparticles, galectin-1 (Au-Gal1) bound with higher affinity to Jurkat cells to promote CD45 clustering and inhibition of its phosphatase activity, resulting in enhancement of apoptosis via caspase-dependent pathways. Au-Gal1 injected intra-articularly into rats with collagen induced arthritis (CIA) promoted apoptosis of CD4+ T cells and reduced pro-inflammatory cytokine levels in the ankle joints as well as ameliorated clinical symptoms of arthritis. These observed therapeutic effects indicate that the multivalent structure of nanoparticle-ligands can regulate the distribution of cell surface receptors and subsequent intracellular signalling, and this may provide new insights into nanoparticle applications.
論文目次 Contents
Abstrate in Chinese…………………………………….......………………III
Abstrate…...…………………………………………….......……………….V
Acknowledgement…………… …………………………..….………….. VII
Contents…………………………………………………..….…………...VIII
Index of Figures………… …………………………….….….…………….XI
Index of Appendixes....................................................................................XII
Abbreviations…………………………………………….………………XIII
Introduction……………………………………………….……..…………..1
Multivalent ligand………………….……………………………....………..1
Galectins……………………………….……………………………....….….2
Galectin-1…..………………………….….…………………………….……3
Nanoparticles in medicine……………..…………………………………….6
Gold nanoparticles…………………….……….…………………………....7
Rheumatoid arthritis………………….……….…………………………….8
Rationals and Specific Aims………….……..…..……..……………..……10

Materials and Methods…………………..……….………………………..11
1. Materials..……………………….……..…….…………………………11
1.1 Plasmids…………………………….….……….…………………….……..11
1.2 Cell lines……………………….…..…………………………………..11
1.3 Animals..…………………………….….…………………………………...11
2. Methods..………..…………………….…………………………….……....11
2.1 Cells……………………………………………………………...………......11
2.2 Rats..………………………………………………………...………………11
2.3 Production of recombinant galectin-1 protein…………………...….12
2.4 Preparation and characterization of galectin-1 conjugated to gold nanoparticle……………………………………………………...12
2.5 Mechanism of binding……………….………………………………..13
2.6 Binding avidity………………………….……………………………..14
2.7 Cell viability.………………………………...………………………...14
2.8 Western blot analysis…………………………………………………14
2.9 CD45 clustering assay………………………………...………………15
2.10 Induction of CIA and delivery of Au-Gal1……………...…………15
2.11 Clinical and radiographic assessment………………………...……16
2.12 Histopathologic and immunohistochemical assessment……...…...16
2.13 Enzyme-linked immunosorbent assay (ELISA)………...…………17
2.14 Statistical analysis……………………………………………………17
Results……………………………………………………………………….18
Binding mechanism of galectin-1 onto gold nanoparticle surface..........18
Au-Gal1 complexes preserved the biological activity of galectin-1……18
Au-Gal1 enhanced T cell apoptosis via a caspase
dependent pathway………………………………………………...……...19
Au-Gal1 increased binding affinity and CD45 clustering…………..….20

Au-Gal1 enhanced CD4+ T cell apoptosis and
ameliorated clinical symptoms of CIA……………….…..……………....21
Discussion……………………………………………………………...……24
The interaction between galectin-1 and gold nanoparticle…………......24
The mechanism of Au-Gal1-induced T cell apoptosis……………..........25
The therapeutic effect of Au-Gal1 on collagen-induced arthritis……...26
Conclusion………………………………………………….…………...…..28
References…………………………………….….…………………...…….29
Figures……………………………………………………………...……….35
Appendixes………………………………………………………………….52
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