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研究生: 黃嘉雯
Jia-Wen Huang
論文名稱: Galectin-1誘導前脂肪細胞分化機制之研究
Mechanism of galectin-1-induced preadipocytes differentiation
指導教授: 黃榮南
Rong-Nan Huang
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度: 92
語文別: 英文
論文頁數: 58
中文關鍵詞: 前脂肪細胞分化
外文關鍵詞: galectin-1(GAL1), preadipocytes, differentiation, two dimensional electrophoresis
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  • 中 文 摘 要
    Galectin-1 (GAL1)是一種?-半乳糖苷結合的蛋白,具有參與細胞貼附、發育及生長調控的功能。已有許多的研究指出GAL1對於促進細胞貼附於細胞外基質及誘導細胞的增生而言,扮演重要的角色,然而有關GAL1影響細胞分化的研究並不多。本研究發現GAL1蛋白與IDM (促分化劑)相仿,具有促進前脂肪細胞分化之作用;然其促進前脂肪細胞分化之機制,則與IDM明顯不同;IDM促進3T3-L1前脂肪細胞分化時,3T3-L1細胞會先進行兩次之細胞分裂,而後進行分化作用,成為脂肪細胞;利用Flow cytometry分析,發現GAL1促進3T3-L1前脂肪細胞分化之過程,並無細胞分裂現象;此外, GAL1蛋白促使C/EBP ?、C/EBP ?、PPAR ?、Adipsin等脂肪細胞形成(adipogenesis)之轉錄因子表現,與IDM之作用亦不相同。另外,利用dominant negative C/EBP 處理時,發現由GAL1所誘導的分化作用並不會受到抑制,此一結果顯示GAL1誘導前脂肪細胞之分化,並不是經由C/EBP之訊號傳遞過程,明顯IDM與之機制不同。除此之外,藉由二維電泳的分析,發現由GAL1所誘導的脂肪細胞分化,許多特異蛋白質的表現,將藉由MALDI-TOF來鑑定這些蛋白質的種類。


    Abstract
    Galectin-1 (GAL1, a b-galactoside-binding protein) functions in cell adhesion, development, and growth regulation, but the mechanism of GAL1-induced cell differentiation remained to be defined. In this study, we showed that coating of GAL1 in culture plates promoted the differentiation of 3T3-L1 preadipocyte, which can be dose-dependently inhibited by GAL1 inhibitor, lactose (50-200 ?g/ml) and TDG (10-30 mM). Fluorescence-activated cell sorting (FACS) analysis showed that GAL1 treatment did not induce mitotic clonal expansion of adipocytes, in which has been thought to be an essential preliminary step of adipocyte differentiation. In addition, expression of dominant negative C/EBP α and C/EBP β showed no effect on GAL1-mediated 3T3-L1 differentiation. These results suggested that GAL1-mediated 3T3-L1 preadipocyte differentiation is likely through C/EBP?- and C/EBP?-independent pathway. Thus, it is evident that the mechanism of GAL1 action on 3T3-L1 preadipocyte differentiation seems distinct from that induced by IDM.

    Contents Introduction…………………………………………………01 Ⅰ. Galectins a. GAL1 b. Biosynthesis of GAL1 c. Function of GAL1 Ⅱ. Adipocyte Differentiation Material and Methods………………………………………...11 1. Cell culture and Differentiation induction 2. SDS-PAGE 3. Western blot 4. Oil Red O Staining 5. Flow cytometry 6. Transient transfection 7. Two-dimensional electrophoresis Results……………………………………………………….15 1. Expression of GAL1 on 3T3-L1 adipocyte 2. Attenuation of GAL1-dependent preadipocytes differentiation by TDG 3. Expression pattern of GAL1 during the differentiation of 3T3-L1 preadipocytes to adipocytes 4. Cell cycle progression in GAL1-induced adipocyte differentiation 5. Expression profiles of adipogenic specific protein in GAL1-induced 3T3-L1 differentiation 6. Signalling pathway involved in GAL1-induced 3T3-L1 Preadipocyte differentiation 7. Two-dimensional electrophoresis analyses of proteins expression in GAL1- or IDM-mediated 3T3-L1preadipocyte differentiation Discussion……………………………………………………..21 References……………………………………………………..27 Figures………………………………………………………...39

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