臺灣博碩士論文加值系統

間葉幹細胞(MSCs)為多效性祖源細胞能分化成多種細胞，例如硬骨母細胞、軟骨母細胞、橫紋肌母細胞、脂肪細胞和神經母細胞等。黏著斑(focal adhesion，FA)為細胞底部與胞外基質連接的結構，也具有傳遞訊息和形成胞外基質的能力。之前的研究顯示間葉幹細胞分化的調控與黏著斑轉為成熟型態有關。雖然已知人工合成的醣皮質類固醇(Dexamethasone，Dex)能誘導間葉幹細胞進行骨分化，但醣皮質類固醇如何調控黏著斑的相關機制我們仍不是很清楚。我們發現醣皮質類固醇能促進GEF-H1移動至黏著使黏著斑穩定和有利於骨分化的進行。為了探討GEF-H1如何經由調節黏著斑的組成來調控肌動蛋白骨架的結構和訊息的傳遞，我們分別取從對照組和GEF-H1 -silencing組分離出來的黏著斑進行蛋白質體分析並探討兩者的黏著斑組成由何差異。我們在間葉幹細胞的黏著斑中辨識出346個蛋白，其中有63%(219)的蛋白會因為GEF-H1的表現而在黏著斑中有顯著得變動。根據蛋白質體分析的結果，我們將重點放在NMIIB並探討NMIIB在GEF-H1在經由調控細胞骨架來驅使間葉幹細胞進行骨分化中扮演什麼角色。

Mesenchymal stem cells (MSCs) are multipotent progenitor cells with abilities to differentiate into multiple cell lineages, such as osteoblasts, chondroblasts, myocytes, adipocytes and neurocytes. The regulation of MSC commitment has been linked to the maturation states of a complex plasma-membrane associated macromolecular organelle, focal adhesion (FA), that connect cells to, as well as transduce signals to and from, the surrounding extracellular matrix (ECM). Although a synergistic steroid, dexamethasone (Dex) can induce MSCs osteogenic differentiation, the underlying mechanism of FA modulation by Dex remains unknown. Here, we found that Dex promotes the recruitment of guanine nucleotide exchange factors H1 (GEF-H1) into FAs that mediates FAs formation, stress fibers orientation, and MSCs osteogenesis. To further determine the mechanism of GEF-H1 in regulating FA formation, the organization of actin cytoskeleton, and specifying MSCs osteogenic commitment, we hypothesize that FA recruitment of GEF-H1, in response to Dex treatment, transduces signals through globally modulating FA composition. To achiev this hypothesis, we performed a proteomics approach to isolate native FAs, identify their protein composition, and compare specific protein abundance in FAs from Dex-treated MSCs with and without GEF-H1 expression. We reproducibly identified 346 FA-associated proteins from Dex-treated MSCs, of which 63% (219) had significant changes in FA abundance in response to GEF-H1 expression. We focused on the non-muscle myosin II heavy chain B (NM IIB), documenting its role in GEF-H1-mediated cytoskeletal organization to drive MSCs osteogenic differentiatio

Contents

Signature Page i
Thesis Approval Form ii
Acknowledgments iii
Chinese Abstract iv
English Abstract v
Chapter 1 Introduction 1
Mensenchymal stem cells differentiation 1
Mesenchymal stem cells osteogenic differentiation 3
Cytoskeletal mechanics in mesenchymal stem cells osteogenic differentiation
5
Guanine nucleotide exchange factor (GEF-H1) 6
Chapter 2 Material and Methods 8
Material 8
Methods 10
Chapter 3 Result 15
Microtubule stabilization influences Dex-stimulated stress fibers formation and FA maturation. 15
GEF-H1 mediates Dex-mediated cytoskeletal architecture. 16
Dexamethasone treatment increases the recruitment of GEF-H1 into FAs 17
GEF-H1 plays crucial in organizing functional FAs. 18
GEF-H1 is required for FA recruitment of NMIIB. 20
GEF-H1 recruits NMIIB to FAs via its DH domain.. 21
NM IIB regulates Dex-induced stress fiber orientation. 22
GEF-H1 plays crucial in Dex-driven MSCs osteogenesis. 23
NMIIB at FA is essential for Dex-driven MSCs osteogenesis. 24
Chapter 4 Discussion 25
GEF-H1 modulates stress fiber alignment through microtubule dynamics. 25
GEF-H1 regulates actin organization not through RhoA activation. 26
GEF-H1 modulate FA composition to alter FA signaling. 26
The interaction between NMIIB and GEF-H1 is essential for stress fiber orientation. 28
GEF-H1 drives osteogenic differentiation through NMIIB staying at FA to regulate stress fiber alignment 28
Conclusion 29
References 30
Figures and Legends 40
Table 1 56

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