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系統識別號 U0026-2108201216584100
論文名稱(中文) 上皮間質化前後在不同勁度與表面型態的基材對細胞遷徙之影響
論文名稱(英文) Cell Migration after Epithelial Mesenchymal Transition on Various Substrate Stiffness and Topography
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 李佳徽
研究生(英文) Chia-Hui Li
學號 P86991193
學位類別 碩士
語文別 英文
論文頁數 61頁
口試委員 指導教授-葉明龍
口試委員-張志涵
口試委員-王士豪
口試委員-艾群
中文關鍵字 上皮間質轉換  正常乳腺上皮細胞  轉化生長因子β1  聚二甲基矽氧烷  微機電製程 
英文關鍵字 Epithelial to mesenchymal transitions (EMT)  Normal marine mammary gland cells (NMuMG)  Transforming growth factor-β1 (TGF-β1)  Polydimethylsiloxane (PDMS)  Micro engineering mechanical system (MEMS) 
學科別分類
中文摘要 上皮間質轉換過程在許多生理及病理上扮演重要的角色。其過程是將極化的上皮細胞轉換成具有移動性的間質細胞,使細胞具有轉移及遷徙的特性。利用生長因子可以在體外誘發上皮細胞的轉換失去極性及細胞骨架重組。本實驗利用控制基材的軟硬度及不同的表面型態,來控制及影響細胞的移動和貼附情形。
本實驗利用常使用為上皮間質轉換的模型正常乳腺上皮細胞(NMuMG),以10:1 及20:1兩種比例混合的矽酮(Silicon elastomer)及熟化劑矽酮樹脂溶液(curing agent)調配出不同軟硬度的聚二甲基矽氧烷(PDMS)為基底材料。其揚氏係數分別為3.70±0.74 及 1.96±0.48 MPa。分別具有兩種不同線寬1μm and 5μm。細胞於不同基材表面貼附24小時後 及生長因子誘發48小時後,連續觀察6小時。利用Matlab分析移動的速率及方向。
兩種細胞均會沿著5μm的溝槽上爬行,間質細胞在大部分的基材上都表現拉長的細胞型態。在大部分的基材型態上,上皮間質轉換後細胞(NMuMGemt)的移動速率比上皮細胞(NMuMG)移動的快。但是在比較硬的1μm 溝槽上上皮細胞移動的速度卻比間質細胞來的快 (p<0.0001)。在軟硬度基材比較上,間質細胞在較軟的基材上移動速度均比在較硬的基材來的快。但在上皮細胞卻只有在1μm
圓柱在較軟的移動速度較快。上皮細胞在較硬的1μm溝槽移動速度最快(0.34±0.05μm/min)而間質細胞(0.34±0.02μm/min)在軟的平面移動速度較快。
綜觀來說,利用基材型態可以利用物理性來影響細胞的移動的速度,間質細胞在基材的軟硬度上是較為敏感的。未來應用在腫瘤生物學的治療及開發藥物上都可利用基材物理性影響來做為應用的工具。
英文摘要 Epithelial to mesenchymal transitions (EMT) is the process that converts polarized epithelial cells into motile mesenchymal cells, and endows cells with invasive and migratory properties. It is known cell-substrate interaction is related to migration velocity and direction. However, the effects of substrate stiffness and topography on migration of cells after EMT are still unknown. We used normal murine mammary gland (NMuMG) as EMT model in vitro to investigate how substrate regulated a series of cellular processes, including spreading, adhesion, and migration.
The polydimethylsiloxane (PDMS) with ratios of silicon elastomer/curing agent concentration at 10:1 and 20:1 were fabricated for soft substrata. The Young’s moduli of PDMS substrata with different ratios were measured by material testing system (MTS) at 3.70±0.74 and 1.96±0.48 MPa, respectively. The surface patterns in the study included flat-top cones and long grooves with line width at 1μm and 5μm. Two types of cells, NMuMG and after EMT (NMuMGemt) with TGF-β1 induction, were seeded on flat PDMS, flat-top cone, or long groove PDMS for 48 hours, to establish stable attachment and EMT. Following a 6 hours migration assay, time-lapsed images were taken by optical microscopy every 5 minutes. The speed and direction of cell migration were calculated by the coordinate of each cell on the image sequence.
Most cells after EMT were faster on all substrata then NMuMG. The highest speeds of NMuMG and NMuMGemt cells were 0.34±0.05 and 0.34±0.02 μm/min obtained on the soft 1 μm groove and soft flat PDMS, respectively. However, the speeds of NMuMG on stiff (p=0.03) 1u grooves were faster than that of NMuMGemt.
In conclusion, this study demonstrated that NMuMG after EMT were more sensitive to the changes of substrate stiffness, and long groove was the best morphology to control cell migration along particular direction.
論文目次 ABSTRACT............... I
摘要....................III
致謝......................................................IV
TABLE OF CONTENTS.........................................V
LIST OF FIGURES........................................ VII
LIST OF TABLES..........................................VIII
CHAPTER 1 INTRODUCTION....................................1
1.1 EMT................................................ 1
1.1.1 TGF-β1-induced EMT............................... 3
1.1.2 Cell Migration Process........................... 4
1.1.3 Adhesion in Cell Migration........................5
1.1.4 Cell Migration after EMT..........................7
1.2 CELL-SUBSTRATE INTERACTION..........................8
1.2.1 Surface Topography................................9
1.2.2 Substrate Stiffness..............................10
1.4 MOTIVATION.........................................13
1.5 PURPOSE............................................13
CHAPTER 2 METHODOLOGY..................................14
2.1 FLOW CHART.........................................14
2.2 CELL CULTURE.......................................15
2.3 FABRICATION OF SILICON WAFER MOLD..................15
2.4 PREPARE PDMS SUBSTRATE.............................16
2.5 MEASUREMENT OF YOUNG’S MODULUS OF PDMS.............17
2.6 PDMS SURFACE MODIFICATION..........................18
2.6.1 Oxygen Plasma....................................18
2.6.2 Coating ECM (Type I collagen)....................18
2.7 CONTACT ANGLE MEASUREMENT..........................20
2.8 MORPHOLOGY OF SPREADING CELL AND PATTERN ON PDMS...21
2.9 CELL MIGRATION ASSAY...............................22
2.10 IMMUNOFLUORESCENT STAINING........................23
2.11 IMAGE ANALYSIS AND STATISTICAL ANALYSIS...........24
CHAPTER 3 RESULTS......................................25
3.1 PDMS MODULUS.......................................25
3.2 SURFACE CHARACTERIZATION...........................26
3.3 ACCURACY OF SURFACE MORPHOLOGY.....................27
3.4 CELL MORPHOLOGY....................................29
3.5 ORGANIZATION AND ALIGNMENT OF Α-SMOOTH MUSCLE ACTIN (Α-SMA) AND FOCAL ADHESIONS (FAS).........................34
3.6 MIGRATION DIRECTION................................38
3.7 MIGRATION SPEED....................................40
3.7.1 Migration Speed Influenced by Substrate Stiffness.40
3.7.2 Cell Migration Speed Influence by Cell Type.......44
3.7.3 Migration Speed Influence by Substrate Topography.47
3.7.4 Migration Speed Influence by Line Width...........48
CHAPTER 4 DISCUSSIONS...................................49
4.1 EFFECT OF SUBSTRATE MORPHOLOGY AND EMT ON NMUMG.....49
4.2 CELL ADHESION AFTER EMT.............................50
4.3 MIGRATION DIRECTION.................................51
4.4 EMT COULD INCREASE CELL MIGRATION...................52
4.5 SUBSTRATE STIFFNESS EFFECT OF CELL MIGRATION........53
4.6 EFFECT OF SUBSTRATE TOPOGRAPHY ON CELL MIGRATION SPEED 54
4.7 EXPERIMENTAL LIMITATIONS............................55
4.8 FUTURE PROSPECTS....................................56
CHAPTER 5 CONCLUSIONS...................................57
REFERENCES..............................................58
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