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系統識別號 U0026-1008201503241200
論文名稱(中文) 物理和生理因子對以原子力顯微鏡為基礎的細胞勁度量測造成的影響
論文名稱(英文) The Influence of Physical and Physiological Cues on Atomic Force Microscopy-Based Cell Stiffness Assessment
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 103
學期 2
出版年 104
研究生(中文) 邱昱瑋
研究生(英文) Yu-Wei Chiou
學號 P88951199
學位類別 博士
語文別 英文
論文頁數 66頁
口試委員 指導教授-葉明龍
口試委員-湯銘哲
口試委員-艾群
口試委員-陳嘉炘
口試委員-邱文泰
口試委員-莊漢聲
中文關鍵字 原子力顯微鏡  肌動蛋白  細胞內骨架  膠原蛋白  細胞力學 
英文關鍵字 Atomic force microscopy (AFM)  Actin filaments  Cytoskeleton  Collagen  Cell mechanics 
學科別分類
中文摘要 原子力顯微鏡除了能提供高解析度的樣本表面形貌外,亦可得到樣本的機械力學特性。利用原子力顯微鏡的接觸、半接觸或非接觸式等操作模式可對樣本做出壓痕、潛變、摩擦係數、相位落後、疲勞等試驗。在壓痕試驗中,藉由得到負載、探針偏折量與探針移動量之間的關係,可獲得樣本的機械力學性質。又因為原子力顯微鏡可以在仿生環境下操作,故能量測各種細胞在一般培養條件下貼附狀態時的表面形貌及機械力學性質。而在眾多機械性質中,細胞彈性被用來泛指細胞在不同條件下的結構強度。本研究主要著重在物理和生理因子們是否會影響以原子力顯微鏡為基礎的細胞彈性量測。
在物理因子方面包含了原子力顯微鏡的探針形狀、負載力量以及原子力顯微鏡使用時的操作溫度。經由實驗結果可知這些因子均對以原子力顯微鏡為基礎的細胞彈性量測產生顯著的影響。使用尖銳的原子力顯微鏡探針所量測出的細胞等效彈性模數會較使用鈍的探針增加近兩倍;當在相同的負載速率下,較高的壓測力量會造成較高的細胞彈性估計量;細胞內的肌動蛋白纖維結構亦會因原子力顯微鏡的操作溫度增加而被瓦解,而導致了細胞勁度的下降。而不論是在正常小鼠纖維母細胞或是將其Ha-RasV12過度表現後的細胞上均發現這些物理因子影響細胞勁度的現象。
在生理因子的影響方面則涵蓋了胎牛血清、細胞外基質、細胞繼代培養的代數、以及細胞密度。從實驗結果發現,胎牛血清和細胞外基質這兩者的重要性在於使正常小鼠纖維母細胞能維持肌動蛋白纖維的完整性而使細胞呈現出較高的細胞彈性。迥異於初代培養細胞,老鼠腎臟前驅細胞在長時間的繼代培養狀況下仍可繼續維持著它的型態和細胞彈性而不至老化。最後,細胞彈性隨著細胞密度上升而增加的情形則僅現於狗的腎小管上皮細胞株之上,在正常小鼠纖維母細胞上則沒有發現類似的結果。
總結來看,本研究結果提供了基礎且重要的定量分析結果使得原子力顯微鏡的使用者可得知在量測細胞彈性時有哪些物理和生理因子會對以原子力顯微鏡為基礎的測量結果產生影響。而使原子力顯微鏡的使用者在操作實驗時能考量到這些生理和物理因子的影響,以減少誤判細胞的機械力學特性的可能。
英文摘要 Atomic force microscopy can provide researchers not only high-resolution surface topography, but also the mechanical properties of samples. By using the contact, the semi-contact, and the non-contact operating model of atomic force microscopy, several experiments such as indentation, creep, friction coefficient, phase lag, fatigue, and so forth can be achieved. In indentation experiment, the mechanical properties of sample can be calculated by obtaining the relations between load, the deflection of cantilever, and the movement of cantilever. In addition, the atomic force microscopy allows sample to be assessed in the pseudo-physiological environment. Hence, the surface morphology and mechanical properties of cells which attached to the surface in normal culturing conditions can be obtained. Amongst all mechanical properties, cell elasticity has been abundantly used to represent the structural stiffness of cells in different conditions. This study investigated whether physical or physiological cues affect cell elasticity in atomic force microscopy-based assessments.
The physical cues include the geometry of the atomic force microscopy tips, the indenting force and the operating temperature of the atomic force microscopy. All of these cues show a significant influence on the cell elasticity assessment. Sharp atomic force microscopy tips create a two-fold increase in the value of the effective Young's modulus (Eeff) relative to that of the blunt tips. Higher indenting force at the same loading rate generates higher estimated cell elasticity. Increasing the operation temperature of the atomic force microscopy leads to decreases in the cell stiffness because the structure of actin filaments becomes disorganized. The physiological cues include the presence of fetal bovine serum or extracellular matrix-coated surfaces, the culture passage number, and the culture density. Both fetal bovine serum and the extracellular matrix are critical for cells to maintain the integrity of actin filaments and consequently exhibit higher elasticity. Unlike primary cells, mouse kidney progenitor cells can be passaged and maintain their morphology and elasticity for a very long period without a senescence phenotype. Finally, cell elasticity increases with increasing culture density only in Madin-Darby canine kidney (MDCK) epithelial cells.
In summary, for researchers who use atomic force microscopy to assess cell elasticity, the results of this study provide basic and significant quantitative results about the effects of physical and physiological cues on the measuring results of atomic force microscopy-based assessment. Eventually, the results of this study help the atomic force microscopy users to consider the effects of these physical and physiological cues, consequently to reduce the possibility of misjudge the mechanical properties of cells.
論文目次 Tables of Contents
ABSTRACT I
中文摘要 III
TABLES OF CONTENTS V
誌謝 VII
GLOSSARY OF ABBREVIATIONS IX
TABLE LIST XI
FIGURE LIST XII
CHAPTER I: INTRODUCTION 1
1.1 THE COMPOSITION AND FUNCTIONS OF CELL ADHESIONS: FROM THE CELL MEMBRANE TO THE CELL NUCLEUS 1
1.2 THE EFFECTS OF CYTOSKELETON ON CELL ELASTICITY 5
1.3 INSTRUMENTS TO STUDY CELL MECHANICS 7
1.4 THE FUNDAMENTALS OF AFM 8
1.5 PURPOSES AND AIMS 13
CHAPTER 2: MATERIALS AND METHODS 15
2.1 CELL LINE AND CELL CULTURE 15
2.2 ADHERENT SUBSTRATE PREPARATION 15
2.3 CULTURING PARAMETERS 16
2.4 AFM SYSTEM 16
2.5 AFM CANTILEVERS AND MEASUREMENT PARAMETERS 16
2.6 TOPOGRAPHIC IMAGES BY AFM 17
2.7 DATA ANALYSIS 17
2.8 IMMUNOFLUORESCENCE STAINING 19
CHAPTER 3: RESULTS 20
3.1 EFFECT OF AFM TIP SHAPES, INDENTING FORCES, AND OPERATING TEMPERATURE ON CELL STIFFNESS 20
3.2 EFFECT OF CULTURING CONDITIONS ON THE CELL STIFFNESS 26
3.3 EFFECT OF THE ADHERING SUBSTRATE ON CELL STIFFNESS 29
3.4 EFFECT OF CULTURE PASSAGE NUMBER ON CELL STIFFNESS 32
3.5 EFFECT OF CULTURE DENSITY ON CELL STIFFNESS 34
3.6 EFFECTS OF EXPERIMENTAL TIME ON CELL ELASTICITY 40
CHAPTER 4: DISCUSSION 42
CHAPTER 5: CONCLUSION 55
CHAPTER 6: LIMITATION AND FUTURE WORK 57
REFERENCES 59
CURRICULUM VITAE 65
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