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研究生:林筱筠
研究生(外文):Hsiao-Yun Lin
論文名稱:細胞外基質誘導人類間葉幹細胞分化成胰島素分泌細胞的機制探討
論文名稱(外文):The mechanism of extracellular matrix involved in mesenchymal stem cells differentiation into insulin-producing cells
指導教授:王盈錦洪士杰
指導教授(外文):Yng-Jinn WangShih-Chieh Hung
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:39
中文關鍵詞:間葉幹細胞胰島素分泌細胞細胞外基質
外文關鍵詞:mesenchymalinsulin producing cellextracellular matrix
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間葉幹細胞 (Mesenchymal stem cells (MSCs)) 由骨髓取得,可以分化成不同細胞且可作為胰島素分泌細胞 (Insulin producing cells (IPCs)) 的來源。β細胞外面的細胞外基質 (Extracellular matrix (ECM))主要為Fibronectin (FN) 及 Laminin (LAM) 兩種。本實驗利用四階段懸浮培養的方式,添加FN及LAM還有IBMX及Nicotiamide將間葉幹細胞誘導為胰島素分泌細胞。實驗結果發現,間葉幹細胞經過分化後本身會表現神經前驅細胞基因Nestin以及胰臟前驅細胞基因Ngn3、Nkx6.1。添加FN或LAM的組別則會大量的增加基因insulin及Glut2的表現在insulin及Glut2的蛋白質表現得到驗證,而細胞質內也有表現α、β、δ granules。添加FN及LAM的細胞經過高濃度葡萄糖的刺激後胰島素的分泌濃度隨之上升。以上實驗證實了添加細胞外基質FN或LAM確實會促進IPC的分化。在探討機制的部份,我們發現了FN及LAM會促進AKT及ERK的表現,意外的我們在添加PD98059 (ERK抑制劑) 及LY294002 (AKT抑制劑) 後,發現了AKT與ERK之間的交互作用。基於以上的實驗可得知,細胞外基質FN及LAM會藉由活化ERK及AKT pathway促進IPC分化。
Multipotential mesenchymal stem cells (MSCs) isolated from bone marrow can differentiate into multiple mesenchymal tissues and can serve as a source of insulin producing cells (IPCs). Fibronectin (FN) and laminin (LAM) are the main extracellular matrices (ECM) around β-islet cells. In this study, we treated MSCs with FN and LAM in a pellet culture and used a four﹣stage differentiation protocol containing neuronal differentiation factor-IBMX and IPC-conversion reagent-nicotinamide to induce MSC to differentiate into IPCs. Achievement of differentiation was evaluated by means of reverse transcription–polymerase chain reaction, real-time RT-PCR, immunostaining, transmission electron microscopy (TEM) and functional analysis. MSCs expressed genes of neural progenitor cells such as Nestin and islet transcription factors, such as Nkx6.1, Ngn3, but expressed limited insulin and Glut2 genes after induction without adding ECM. Adding FN and LAM enhanced pancreatic differentiation with increases in insulin and Glut2 gene expressions, in proinsulin and insulin protein levels, and in cytoplasmic granules including α, β, and δ granules. Further, IPCs from induction with ECM secreted more insulin in response to elevated glucose concentration, compared to induction without ECM. The high glucose-induced secretion of insulin was regulated by reagents that increase cyclic AMP production or modify calcium influx. We then explored the underlying mechanism and found that AKT and ERK were activated by FN and LAM. Blocking ERK and AKT activation by adding PD98059 (MEK specific inhibitor) and LY294002 (PI3K specific inhibitor) induced the activation of AKT and ERK, respectively and also induced the expression of insulin and Glut2, suggesting the crosstalk of these pathways. Blocking both ERK and AKT by adding PD98059 and LY294002 at the same time inhibited the expression of insulin and Glut2, suggesting IPC differentiation enhanced by ECM is ERK and AKT dependent. Our data demonstrate that FN and LAM promotes IPCs derived from MSCs by activating ERK and AKT.
Abstract 1
摘要 3
Chapter 1 Introduction 4
1-1 Diabetes Mellitus 4
1-1.1 Complication 5
1-1.2 Treatments 6
1-2 Pancreas 7
1-3 Insulin 7
1-3.1 Structure 8
1-3.2 Synthesis 9
1-3.3 Release 10
1-4 Human Mesenchymal Stem Cells 11
1-5 Extracellular Matrix 12
1-5.1 Fibronectin 12
1-4.2 Laminin 13
1-4.3 Integrin 14
Chapter 2 Materials and methods 15
2-1 Cell Culture 15
2-2 Insulin producing cell differentiation 15
2-3 RT-PCR and quantitative RT-PCR 16
2-4 Immunohistochemistry 17
2-5 Transmission ElectronMicroscopy 17
2-6 In vitro insulin release assay 17
2-7 Western blotting 18
Chapter 3 Result 19
3-1 Adding of ECM enhances differentiation of 3A6-hMSCs into insulin producing cells 19
3-2 Differentiated of 3A6-hMSCs expresses proinsulin and insulin 19
3-2 LAM increases insulin release by stage IV cells 20
3-3 ECM enhances IPC differentiation by activating ERK and AKT  21
3-4 PI3 Kinase and MEK 1 inhibitor blocks ECM-mediated activation of AKT and ERK to decrease IPC differentiation 22
3-5 Activation of AKT and ERK to enhance IPC differentiation by hMSCs is integrin and FAK-independent 23
Chapter 4 Discussion 24
Chapter 5 Figure 26
References 43
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