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研究生:高偉倫
研究生(外文):Wei-Lun Kao
論文名稱:利用非標定方法探討表面電位對細胞貼附行為之影響
論文名稱(外文):Investigating the Effect of Surface Potential on Cell Adhesion Behavior by Label-free Methods
指導教授:薛景中
指導教授(外文):Jing-Jong Shyue
口試委員:陳培菱羅世強虞邦英王榮輝
口試日期:2018-12-11
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:材料科學與工程學研究所
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:107
語文別:英文
論文頁數:130
中文關鍵詞:細胞貼附自組裝單層膜表面電位石英振盪微天平飛行時間式二次離子質譜儀主成分分析法
DOI:10.6342/NTU201804370
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於探討材料表面性質對細胞行為的影響時,細胞的貼附行為扮演重要的角色。自組裝單層分子可提供簡單且富變化性的表面性質調控,因此適合來探討表面性質對於細胞貼附行為的影響。藉由同時沉積不同比例且具相反電性的官能基於金基材,可調控一系列漸變的表面電位。於之前的研究中,便已探討纖維母細胞以及上皮細胞於此系列表面的細胞密度以及形貌上的差異。這兩種細胞的細胞密度均隨表面電位增加而增加,然形貌上則對表面電位變化而有不同的漸變反應。
石英振盪微天平加裝能量消散偵測器(Quartz Crystal Microbalance with Dissipation Monitor, QCM-D)具有其即時偵測黏彈特性優勢,搭配光學顯微鏡的使用,此表面敏感技術近年應用於即時偵測細胞與表面的交互作用時的黏彈特性以及光學變化。本研究便利用此方法檢測纖維母細胞以及上皮細胞於不同表面電位貼附時,細胞、細胞間質蛋白以及修飾表面之間交互作用的即時變化。同時,用免疫螢光染色法以確認其於貼附後的黏接點分布,於四小時的觀察中,纖維母細胞傾向於貼附展開在正電表面,隨著表面電位的降低,細胞展開程度下降且移動性增加。根據其形貌上的差異,分別使用膜震盪以及耦合震盪模型解釋細胞貼附於偏正以及偏負的表面時QCM-D的訊號變化。纖維母細胞於偏正電表面貼附時,可藉由表面正電荷以及細胞膜間的靜電吸引於表面快速的貼附並展開,其貼附過程僅需少量間質蛋白參與呈現而單一階段。另一方面,細胞於偏負表面貼附時其QCM-D訊號呈現三階段。首先,由於其靜電排斥的影響,細胞不易於表面貼附展開而耦合震盪效應明顯。然此負電表面亦加強間質蛋白於表面吸附,因此間質蛋白的吸附主導於第二階段時的震盪行為,且細胞可藉由間質蛋白的協助於第三階段貼附於表面。由此可知,當表面電位逐漸由正轉為強負電表面時,細胞貼附機制則從表面靜電吸引轉為間質蛋白輔助機制。對於上皮細胞而言,其亦易於偏正電表面展開,而在偏負表面電位則保持移動並團聚。上皮細胞於偏正表面貼附時呈現三階段。首先,藉由表面靜電吸引的影響,其可快速貼附及展開。然由於上皮細胞有均一化的傾向,已經展開的細胞則於第二階段回縮其偽足。於第三階段,改變形狀的上皮細胞則進行間質蛋白的重排使其呈成熟結構。其貼附行為於偏負電表面貼附時則呈現四階段。受限於靜電排斥力的影響,細胞於第一相不易貼附展開,僅可偵測到間質蛋白的吸附影響。然藉由間質蛋白的協助下,細胞方可於第二階段貼附。已貼附的細胞則於第三階段進行間質蛋白的重排。最後,細胞於第四階段時減少細胞與表面交互作用,並加強細胞間交互作用而團聚。總結當表面電位下降時,上皮細胞的貼附行為亦從細胞與表面直接的靜電交互作用轉變為細胞、間質蛋白與表面的複合結構,而其形貌則均具均一化行為。
經過動態觀察可知,表面電位可由靜電交互作用改變上皮細胞以及纖維母細胞的貼附機制。由於細胞所分泌的間質蛋白(Cell Secreted Matrix, CSM)可反映出細胞貼附於表面時的物化反應,為釐清其對間質蛋白相關的貼附機制影響,接下來則針對表面電性對上皮以及纖維母細胞於修飾表面上貼附時CSM的形貌以及特性探討。經過四小時以及二十四小時細胞貼附於修飾表面並去除後,以飛行時間式二次離子質譜儀 (ToF-SIMS) 作為非標定方法分析CSM表面,而其質譜藉由主成分分析法(Principle Component Analysis, PCA)比對與參考間質蛋白之相似性。免疫染色法則用以觀察細胞分泌行為以及去除細胞後非標定法對CSM定性結果的對照。其結果可知,非標定以及染色法對CSM成分得到一致的結果。表面電性對上皮細胞於貼附時期的CSM的成分以及形貌影響不大。經過增生階段後,雖然CSM於正負電性表面組成類似,但由於CSM的不連續,細胞仍能與表面交互作用,其於帶正電表面形成單層結構而於帶負電表面形成聚集結構。另一方面,表面電性對纖維母細胞的影響較為顯著。無論於貼附或增生階段,間質蛋白於正電表面較集中於細胞核,而於帶負電表面分則均勻分布。然而於貼附階段CSM的組成中可以發現,纖維母細胞分泌較高比例的層粘連蛋白於負電表面。此組成的差異在經過增生階段後便消失。
Cell adhesion is crucial to cell behaviors including survival, growth and differentiation. Self-assembled monolayers (SAMs) provide a convenient and versatile means of modifying surface properties to study how environmental cues affect the cell adhesion process. Serial ζ-potential surfaces can be realized by introducing various ratios of oppositely charged functional groups on a gold surface. Static cell density, morphology and proliferation for NIH3T3 fibroblasts and HEK293T epithelial cells on these serial surfaces have been investigated. These two kinds of cells showed the same tendency in cell densities but different morphological responses to the ζ-potential value.
Quartz crystal microbalance with dissipation monitoring (QCM-D) has advantages for examining real-time viscoelastic changes on surfaces. This surface-sensitive technique can be applied in cell adhesion studies to investigate the cell-surface interactions. Combining an optical microscope with the QCM-D system, in situ and real-time cell morphology and corresponding viscoelastic changes can be obtained. In this work, cell-surface, extracellular matrix (ECM)-surface and cell-ECM interaction for NIH3T3 and HEK293T cells on these serial ζ-potential surfaces were examined. The effect of ζ-potential on focal adhesion was also characterized by immunostaining. For NIH3T3 cells, the morphological results indicated that cells were prone to spread on surfaces of more positive potential, while more negative potentials led to more cell movement on the surface. When cells adhered on more positive charge surfaces, soft and elastic cell bodies with less ECM layer on the surfaces can be sensed by QCM-D. Cells adhere to the surface and spread more quickly owing to electrostatic attraction. The shift in resonant frequency and energy dissipation of the quartz substrate can be described using a film resonance model, and a single-phase adhesion process was observed. On the other hand, for surfaces of more negative potential, round cells were observed and behave similar to coupled oscillators on the QCM-D sensor. Furthermore, three phases were observed during the cell adhesion process. Initially, round cells interact with the surface weakly with a point contact due to the repulsive interaction between negatively charged cell membranes and the surface. For higher magnitude of surface charge, more rigid ECM was deposited on the surfaces in the second phase. Finally, cells then adhered on the surface through the ECM layer. In other words, the mechanism of cell adhesion changed from an electrostatic cell-surface interaction to a cell-ECM-surface composite. For HEK293T cells, cells were also prone to spread and form more focal adhesion sites on surfaces with more positive charge (more NH2 groups) but aggregated and remained highly mobile on surfaces with more negative charge (more COOH groups). On NH2-rich surfaces, cells underwent three-phase kinetics during the adhesion process. Initially, for attractive interaction between NH2 groups and cell membrane, cells adhered and spread quickly on the NH2-rich surfaces with less ECM sensed. The epithelial cells then shrank their filopodia in the second phase to normalize their size. In the final phase, cells underwent ECM remodeling and formed matured ECM. On COOH-rich surfaces, four phases were identified during the cell adhesion process. Initially, due to electrostatic repulsion between the negatively charged cell membrane and surfaces, direct cell adhesion and spreading were restricted. However, ECM was quickly deposited. In the second phase, cells adhered on and interacted with the surface through the ECM layer. In the third phase, cells underwent ECM remodeling, and additional ECM was deposited on the surfaces. Finally, instead of cell-surface interactions, the cells aggregated to form cell-cell junctions.
From the dynamic observation, it has been known that these charged surfaces had electrostatic influence on adhesion mechanism of HEK293T epithelial and NIH3T3 fibroblast cells. However, the ECM-associated adhesion mechanisms were still not clear. Cell secreted matrices (CSM), where extracellular matrix proteins (ECM) deposited by monolayer cells, can help us to understand cell-ECM interactions and find the cues of the cell fate on material surfaces. The composition and the morphology of CSM were examined to investigate the effect of surface charge on epithelial and fibroblast cells adhering on these SAM-modified surfaces. After incubation, cells were removed and time-of-flight secondary ion mass spectrometry (ToF-SIMS) was used to generate spectra for each CSM surfaces. Principle component analysis (PCA) was used to extract the extract the trend of similarly between CSM and referential ECM proteins. Immuno-stained cellular images visualize ECM secreting activities and the same staining method was also applied on decellularized CSM sample to verify ToF-SIMS results. For epithelial cells, surface charge has minor effect in morphology and CSM composition in adhesion phase. Although CSM compositions were similar in proliferation phase, the discontinuity of CSM allowed the surface charge affect the cells adhere as monolayer on NH2 surface while aggregate on COOH surface. For fibroblast cells, surface charge affect cells distribute ECM proteins more localized around nuclei on NH2 surface and uniformly on COOH surfaces in adhesion and proliferation phase. In adhesion phase, higher laminin loading was found in CSM composition on COOH surface than on NH2 surface to alleviate effect of surface charge. However, CSM compositions were surface independent after addition incubation time.
摘要 I
ABSTRACT III
CONTENT VI
FIGURE CAPTIONS X
TABLE CAPTIONS XV
CHAPTER 1 INTRODUCTION 1
1.1 Cell Adhesion 1
1.1.1 Role of Cell Adhesion in Cell Fate 1
1.1.2 Effect of Surface Properties to Cell Adhesion 1
1.2 Self-Assembled Monolayers (SAMs) 6
1.2.1 Self-Assembled Monolayers (SAMs) 6
1.2.2 Binary SAM Modified Surfaces 7
1.2.2.1 Surface Wettability 7
1.2.2.2 Serial Surface Potential Surfaces 8
1.3 Biological Applications of Binary SAM Modified Surface 11
1.3.1 Effect of Surface Potential on Biomolecular Adsorption 11
1.3.2 Effect of Surface Potential on Cell Adhesion and Proliferation 14
1.3.2.1 Effect of Surface Potential on Cellular Densities 14
1.3.2.2 Effect of Surface Potential on Cellular Morphology 16
1.4 Quartz Crystal Microbalance with Dissipation Monitor (QCM-D) 17
1.4.1 Sauerbrey Equation35 17
1.4.2 Dissipation Monitor 18
1.4.3 D-f Plot 19
1.4.4 Sensing Depth 19
1.4.5 Missing Mass Effect 20
1.4.5.1 Film Resonance 23
1.4.5.2 Coupled-Oscillation 26
1.4.6 Using QCM-D as a Biosensor 29
1.4.6.1 Typical Δf and ΔD Response during Cell Adhesion13 30
1.4.6.2 Revealing Phase Transition of Cell Adhesion from D-f Plot59 31
1.4.6.3 Cell Retraction62 33
1.4.6.4 Composite Oscillation during Bacteria Adhesion 35
1.5 Time of Flight Secondary Ion Mass Spectrometry (ToF-SIMS) 38
1.5.1 Principle of ToF-SIMS 38
1.5.2 ToF-SIMS Application in Biological Studies 38
1.5.3 ToF-SIMS Data Analysis with Principle Component Analysis (PCA) 40
1.5.4 PCA Application in Biological Studies 44
1.6 Motivation 46
1.6.1 Effect of Surface Potential on the Kinetics of Cell Adhesion Behaviors Using QCM-D 46
1.6.2 CSM Characterization by Label-free and Staining Methods 47
CHAPTER 2 MATERIALS AND METHODS 48
2.1 Binary SAM-Modified Gold Subtract 48
2.2 Characterization of Serial SAM-modified Surfaces 48
2.3 Cell Culture 49
2.3.1 NIH3T3 Fibroblast Cell 49
2.3.2 HEK293T Epithelial Cell 49
2.4 Cell adhesion behavior observation 49
2.4.1 Cytoskeleton and Focal Adhesion Staining 49
2.4.2 Simultaneous QCM-D and OM Observation 50
2.4.3 SEM Observation 52
2.5 CSM Characterization 52
2.5.1 Preparation of Referential ECM Proteins 52
2.5.2 CSM Production 52
2.5.3 ToF-SIMS 53
2.5.4 PCA 54
2.5.5 Immuno-staining for ECM Proteins 55
CHAPTER 3 RESULTS AND DISCUSSION 57
3.1 Effect of Surface Potential on the Kinetics of Cell Adhesion Behaviors Using QCM-D 57
3.1.1 NIH3T3 Fibroblast Cell Adhesion Behavior 57
3.1.1.1 Focal Adhesion and Morphology 57
3.1.1.2 Real-time Observation of the Cell Adhesion Process in QCM-D 61
3.1.1.3 Effect of Surface Potential on the Change of Viscoelastic Properties during Cell Adhesion 66
3.1.2 HEK293T Epithelial Cell Adhesion Behavior 75
3.1.2.1 Real-time Morphology Observations 75
3.1.2.2 Focal Adhesion of HEK293T Cells after the Adhesion Process 76
3.1.2.3 HEK293T Cell Adhesion Behaviors on NH2-rich Surfaces 78
3.1.2.4 HEK293T Cell Adhesion Behaviors on COOH-rich Surfaces 87
3.2 CSM Characterization by Label-free and Staining Methods 96
3.2.1 PCA classification 96
3.2.2 Classification of proteins deposited from serum 101
3.2.3 Characterization of CSM from HEK293T Epithelial Cells 103
3.2.4 Characterization of CSM from NIH3T3 Fibroblast Cells 111
CHAPTER 4 CONCLUSION 120
4.1 Effect of Surface Potential on the Kinetics of Cell Adhesion Behaviors Using QCM-D 120
4.1.1 NIH3T3 Fibroblast Cell Adhesion Behavior 120
4.1.2 HEK293T Epithelial Cell Adhesion Behavior 121
4.2 CSM Characterization for Epithelial and Fibroblast Cells 122
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