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研究生:林佳屏
研究生(外文):Chia-Ping Lin
論文名稱:藉由限制粘著斑的行成大小調控細胞的運動
論文名稱(外文):Modulations of cell locomotion by restricting the size of focal contact
指導教授:林奇宏林奇宏引用關係
指導教授(外文):Chi-Hung Lin
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:42
中文關鍵詞:粘著斑細胞基質細胞運動
外文關鍵詞:focal adhesionpatterned substratepaxillinFAKY-27632EGF
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粘著班是一種由許多大分子所構成的特殊複合體,在細胞中作為細胞骨架以及細胞外基質連結的橋樑,同時也是整合細胞內部生化訊息的中心,能夠號召多種訊息蛋白調控細胞的活動,例如: 細胞的生長運動、增殖、生存、分化、細胞型態以及細胞凋零。由於不同大小和密度的細胞外基質可引發不同程度的整合蛋白群集,造成下游信息有不同的結果。所以我們利用實驗室已建立的微接觸式印刷技術,印製微米或是次微米等級之線型陣列的圖案生長基質,進而限制細胞粘著班的形成程度,並且探討微米及次微米的細胞外基質如何影響細胞的型態、運動模式和粘著斑的組成。先前的研究發現,表皮生長因子和整合蛋白都能夠藉由活化FAK,來調節下游的訊息傳遞進而促進細胞運動,於是我們給予表皮生長因子的刺激之後,大鼠膀胱表皮細胞便能夠整合由次微米細胞外基質所造成的細胞內紊亂的訊息,並且改善細胞的運動模式。此外,我們也藉由藥物Y-27632的處理來調控粘著斑的形成,Y-27632是ROCK的抗化劑,可以抑制細胞內Rho small GTPase的訊息,藉此抑制了粘著斑的成熟轉化過程。實驗中,我們發現給予Y-27632的大鼠膀胱表皮細胞所形成的粘著斑比較小且較不穩定,並且細胞爬行速度會比較快。且不受到線型陣列的圖案生長基質所拘束。
Focal adhesions are specific types of large macromolecular complexes which serve as a mechanical linkage between the extracellular matrix (ECM) and the cytoskeleton of cells. Focal adhesions work as a biochemical signaling hub which recruits numerous signaling proteins to regulate cellular activities such as cell growth, proliferation, survival, differentiation, morphology, and cell death. Here, we apply micro/sub-micro ECM pattern technology to restrict the size of formation of focal adhesions and study how the physical alterations of ECM substrate affect cell morphogenesis, locomotion and the composition of focal adhesions. It bases on the notion that different sizes and densities of ECM can induce different levels of integrin clustering that in turn, trigger different downstream signalling events. As previous research, epidermal growth factors (EGF) enhance the signalling of focal adhesion kinase (FAK) which shares the same route of downstream integrin signal to promote cell migration. Our results reveal that NBT-II cells can integrate the disordered downstream signaling which is obtained from the sub-micron ECM pattern, and ameliorate the velocity of cell migration after EGF treatment. A pharmacological modulation of focal adhesions is treated cells with Y27632. Y27632 is a sort of ROCK inhibitor, which prohibits focal adhesions from being matured via blocking intracellular Rho small GTPase signaling, the downstream signaling of focal adhesions. We find that NBT-II cells supplied with Y-27632 possess smaller and more unstable focal adhesions than control cells. Besides, the velocity of cell migration is faster in Y-27632 treated NBT-II cells above the micron ECM pattern.
Contents
Signature Page…………………………………………………...…..…….i
Thesis Approval Form……………………………………………..………ii Acknowledgments………………...……………………………...……….iii
Chinese Abstract………………………………………………...…….…..iv
English Abstract………………………………...………….………………v
Table of Contents…………………..…………………………….……….vii
List of Figures………………………….……………………………..…..viii
Chapter 1 Introduction…………………………………….………………1
1.1 Extracellular matrix(ECM), integrin and focal adhesion………1
1.2 The steps of cell migration and Nara bladder tumor cells(NBT-II)…………………………………………………..…. 2
1.3 The function of Rho associated protein kinase (ROCK) in cell locomotion……………………………...…………………………4
1.4 The function of focal adhesion kinase, FAK in cell locomotion…………………………………………………………5
Chapter 2 Materials and methods..……………………………….……...7
2.1 Cell culture…………………..............……………….................7
2.2 Plasmid transfection………………………………...…………...7
2.3 Reagent..…………………………………………………….……8
2.4 Coverslips preparation………………..…………...…………….8
2.5 Micro-contact printing techniques…..……….…...……………..8
2.6 Immunofluorescence staining………….………………………..9
2.7 Cellular activity by time-lapse recording…………...…………10
Chapter 3 Result…………………………………………………...….….11
3.1 The micron patterned substrates guide the cell morphogenesis to stretch and the sub-micron patterned substrates conduct the cell to a spherical form…………………………….…………………………...……11
3.2 The substrate patterns cause diverse migration behaviors………………………………………………………...11
3.3 The micron- patterned substrates induce cells to generate mature and stable focal adhesions, and the sub-micron pattern substrates restrict the maturation of focal adhesions………………….………………………………...…..12


3.4 Cells grown on sub-micron patterned substrates generate multiple protrusions at leading edge and trigger depressed polarization than cells grown on homogeneous and the micro patterned substrates…………………………………………....14
3.5 The signal of cell migration can be improved by enhancing FAK phosphorylation………..…………………………………………15
3.6 Restrained the maturation of focal adhesions can modify the migration behavior which induced by micron patterned substrate…………….....……..…………………………………..16
Chapter 4 Discussion……..……………………...………………….18
Chapter 5 References………………………………………...……..21
Chapter 6 Figures……………………….……………………………26
Figure 1……………………………………………...…………..26
Figure 2……………………………………………...…………..27
Figure 3……………………………………………………….....28
Figure 4………………………………………...………………..30
Figure 5………………………………………...………………..34
Figure 6……………………………………………...…………..36
Figure 7…………………………………………………………..40

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