博碩士論文 92224003 詳細資訊




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姓名 胡明惠(Ming-Hui Hu)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 Galectin-1誘導纖維母細胞於幾丁聚醣膜 上增生之訊號傳遞機制研究
(Signaling mechanism of Galectin-1 induced 3T3 cell proliferation on chitosan membrane )
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摘要(中) 幾丁聚醣是一種天然無毒性的高分子,具有生物可分解性及生物相容性。近幾年來,幾丁聚醣所製成的薄膜已在臨床醫學上作為生物敷料。而Galectin-1(GAL1)是一種β半乳糖苷結合的蛋白質,在於調節細胞的貼附、發育與調控細胞的生長。本實驗室先前的研究指出: GAL1可以促進3T3 (纖維母細胞)在幾丁聚醣膜上增生。因此本論文中進一步探討GAL1誘使3T3細胞在幾丁聚醣膜上增生的機制。從實驗結果得知,GAL1對於3T3細胞的貼附及增生扮演很重要的角色。為了分析細胞增生的訊號傳遞機制,將3T3細胞處理Genistein(酪胺酸激活酶) 抑制劑與Na3VO4
(酪胺酸去磷酸酶抑制劑),並將細胞培養於有GAL1吸附的幾丁聚醣膜上。結果顯示Na3VO4可以協力促進GAL1誘發3T3細胞於幾丁聚醣膜上的增生。然而,細胞單獨處理Genistein 或Na3VO4 對於細胞增生並無明顯差異。另外,以免疫沉澱法測定可得知GAL1誘使3T3細胞增生是透
過cdc2激活酶的活性增加所導致。此外,我們實驗也證實GAL1促使細胞增生是藉由ERK1/2訊號傳遞路徑所調控。綜合本論文實驗結果,我們證實GAL1誘使3T3細胞在幾丁聚醣膜增生的機制是透過ERK1/2的訊號傳遞路徑及cdc2激活酶的活化。
摘要(英) Chitosan is considered to be a very promising biopolymer for various biomedical and pharmaceutical uses because of its nontoxic and
biocompatible natures. In clinical medicine, chitosan membrane has been used as a biological dressing. Galectin-1 (GAL1), a β-galactoside-binding protein, functions in cell adhesion, development, and growth regulation. Previous study in this laboratory have shown that GAL1 support 3T3 proliferation on chitosan membrane. In this study, we further investigated the mechanism of GAL1-induced 3T3 cell proliferation on chitosan membrane. The results show that GAL1 play an important role in enhancing 3T3 cell adhesion and proliferation on the surface of chitosan membrane. To better characterize a plausible signaling pathway, the 3T3 cells were pre-treated with various kinase or phosphatase inhibitors and cultured in a GAL1-coated chitosan membrane. The results show that Na3VO4 (tyrosine phosphatase inhibitor) can synergistically enhance
GAL1-induced 3T3 cell proliferation on chitosan membrane. In contrast,genistein (tyrosine kinase inhibitor) and Na3VO4 had no dramatic effect on
the 3T3 seeded on culture plates. Moreover, immunoprecipitation assay indicates that cdc2 kinase activity is significantly enhanced in 3T3 cells growing on GAL1-coated culture plates. The current results also suggest that GAL1 induces 3T3 cells proliferation through the ERK1/2 signal
pathway. Altogether, we proposed that the signal mechanism of GAL1-induced 3T3 proliferation on chitosan membrane was mediated through ERK1/2 and cdc2 kinase.
關鍵字(中) ★ 幾丁聚醣膜
★ 組織工程
★ 細胞增生
★ 訊號傳遞
關鍵字(英) ★ chitosan
★ tissue engineering
★ 3T3 cell
★ Galectin-1
論文目次 LIST OF CONTENTS ………………………………………I
LIST OF FIGURES ………………………………………IV
ABBREVIATION ………………………………………… VI
CHAPTER 1 INTRODUCTION ………………………………1
1. 1 Tissue engineering …………………………… 1
1. 2 Galectin-1 ……………………………………… 2
1. 3 Chitin and Chitosan ……………………………4
1. 4 Protein tyrosine phosphorylation ………… 7
1. 5 Mitogen-activated protein kinase (MAPK) cascade ………………………………………………… 8
1. 6 Cell cycle and cyclin B-CDK1 …………………………………………………………… 9
1. 7 Preliminary studies …………………………………………………………… 10
1. 8 Specific aim …………………………………… 11
CHAPTER 2 MATERIAS AND METHODS …………………………………………………………… 13
2. 1 Cell culture …………………………………… 13
2. 2 GAL1 protein purification ……………………13
2. 3 Coating of chitosan membrane with GAL1 proteins ……………………………………………… 14
2. 4 Cell attachment and proliferation on chitosan membrane …………………………………… 15
2. 5 Cell proliferation assay …………………………………………………………… 15
2. 6 Fluorescence-Activated Cell Sorter (FACS) analysis …………………………………………………16
2. 7 Western blot analysis …………………………17
2. 7. 1 Protein assay and Quantification …………………………………………………………… 17
2. 7. 2 Gel electrophoresis ……………………… 17
2. 7. 3 Western blot analysis………………………18
2. 8 Immunoprecipitation and CDC2 kinase assay …………………………………………………………… 18
2. 9 Fluorescence histochemistry analysis …………………………………………………………… 19
CHAPTER 3 RESULTS …………………………………… 21
3. 1 Effect of exogenous GAL1 on cell growth on chitosan membranes ………………………………… 21
3. 2 Immunohistochemical localization of GAL1 in 3T3 cells cultured on chitosan membranes …………………………………………………… …… 22
3. 3 Effects of Genistein and Na3VO4 on the growth of 3T3 cells ………………………………… 23
3. 4 Effects of Na3VO4 on GAL1-induced 3T3 cells growth on culture plates ………………………… 24
3. 5 Na3VO4 potentiates GAL1-induced 3T3 cell proliferation on chitosan membrane …………… 24
3. 6 Na3VO4 potentiates GAL1-induced 3T3 cell on G2/M progression …………………………………… 25
3. 7 Effect of Na3VO4 on GAL1-induced cyclin-B and cdc2 protein expression ……………………… 25
3. 8 Effects of Na3VO4 on GAL1-induced cdc2 activity in 3T3 cells……………………………… 26
3. 9 Signaling pathway involved in GAL1-induced 3T3 cells proliferation ………………………… 27
CHAPTER 4 DISCUSSION………………………………… 29
CHAPTER 5 REFERENCE………………………………… 49
APPENDIX ……………………………………………… 57
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指導教授 黃榮南(Rong-Nan Huang) 審核日期 2005-7-23
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