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研究生:葉衍陞
研究生(外文):Yen-Sheng Yeh
論文名稱:基質硬度、靜水壓、滲透壓對細胞移動和型態之影響
論文名稱(外文):Effects of Substrate Stiffness, Hydrostatic Pressure, and Osmolarity on Cell Migration and Morphology
指導教授:郭柏齡郭柏齡引用關係
口試委員:陳淑靜李超煌
口試日期:2013-07-29
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生醫電子與資訊學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:英文
論文頁數:55
中文關鍵詞:靜水壓細胞外基質軟硬度細胞遷移微流道滲透壓
外文關鍵詞:hydrostatic pressureextracellular matrixstiffnesscell migrationmicrofluidicsosmolarity
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細胞在生物體內的生長發育、分化和移動除了受到生物化學刺激之外,物理性刺激如細胞外基質硬度以及滲透壓亦對細胞行為有很大影響。在我們的實驗當中,我們將H9C2肌肉母細胞培養在不同硬度細胞外基質上並且分成對照組和水壓組來探討此肌肉母細胞同時受細胞外基質軟硬度和靜水壓的刺激對細胞分化和細胞型態的影響。另一方面,我們也探討T細胞在二維和三維細胞培養環境微流道中在均勻和梯度滲透壓的情況下,細胞的遷移是否會受到影響。
我們使用聚丙烯醯胺(Polyacrylamide, PA)水膠來當作細胞外基質材料,因為PA水膠的硬度能夠透過調配不同比例的acrylamide 和bis-acrylamide 來製作出不同軟硬度的水膠。H9C2細胞培養於蛋白質fibronectin 附著的水膠表面再分成控制組和10公分靜水壓組並培養48小時後,觀測細胞分化採轉譯因子MyoD表現量和細胞型態。實驗結果顯示,MyoD的表現量在水壓組皆高於控制組,而細胞面積在經過水壓刺激後亦會較控制組來的大,但細胞長寬比則較無明顯差異。因此,靜水壓可能為促進H9C2細胞分化和增加細胞面積的其中一種刺激因素。
在細胞遷移實驗方面,我們將T細胞培養於三種不同滲透壓培養液內(260、337、以及415 mOsm)並計算細胞移動速度。初步實驗結果顯示T細胞在低滲透壓的細胞培養液中,其移動速度會較慢。此外,我們亦製作出兩種微流道來產生滲透壓梯度以觀察T細胞是否會受到滲透壓梯度刺激而影響細胞遷移行為。此兩種裝置分別為二維細胞培養環境的agarose-based微流道和三維細胞培養環境的collagen-filled微流道。滲透壓梯度於三維細胞培養環境微流道能產生約0.031mM/μm的滲透壓梯度,而T細胞在這兩種流道裝置內分別給予均勻和梯度滲透壓環境來觀察細胞的遷移行為。在有滲透壓濃度梯度的環境中並無觀察到細胞會有特定的遷移趨向性。


The cell growth, proliferation, differentiation, and migration are affected by biochemical and biophysical cues, such as the stiffness of extracellular matrix (ECM) and osmolarity. In this work, we studied the effects of hydrostatic pressure and substrate stiffness on the differentiation and morphology of H9C2 myoblast. We also investigated the effects of homogeneous and gradient osmolarity on T cell migration using 2D and 3D microfluidic devices. Polyacrylamide (PA) hydrogel was used as cell culturing substrates and the stiffness of the PA gel was modulated by mixing different ratios of acrylamide to bis-acrylamide. The cell size, aspect ratio, and expression of transcription factor MyoD were measured after culturing the H9C2 cell on fibronectin coated PA gel with and without the presence of 10 cm hydrostatic pressure for 48 hours. The results reveal that the 10-cm-hydrostatic- pressure exposure increased the MyoD expression and cell size, but resulted in insignificant shape change when compared with cells cultured without presence of the pressure. These findings indicate that the hydrostatic pressure promotes the MyoD expression and increases the cell area. As for the cell migration studies, we cultured QL-9 T cells in medium of osmolarity 260, 337, and 415 mOsm. Live cell images were acquired and analyzing the images revealed that the hypotonic solution significantly decreased the migration speed of the T cells. Two microfluidic-based devices were herein designed for studying the cell migration in response to osmotic gradient: one was a three-chambers design made of agarose for studying the cell migration in a 2D plane; another was a two-chambers device with a collagen-filled tunnel in between for investigating cell movement in a 3D space. The osmotic gradient in the collagen-filled tunnel was about 0.031mOsm/μm. Preliminary results showed that such an osmotic gradient did not evoke gradient-directed movement in T cells.

口試委員會審定書 #
誌謝 i
中文摘要 ii
ABSTRACT iii
CONTENTS v
LIST OF FIGURES viii
LIST OF TABLES xi
Chapter 1 Introduction 1
1.1 Hydrostatic Pressure and ECM Stiffness influence on H9C2 Cell Morphology and Differentiation 1
1.1.1 The Stiffness of Extracellular Matrix 2
1.1.2 Hydrostatic Pressure 4
1.1.3 Previous Study in our Lab 5
1.1.4 Purpose of the Study 8
1.2 Osmolarity Influences on T Cell Migration 9
1.2.1 Osmolarity 9
1.2.2 Aquaporin1 (AQP1) 10
1.2.3 Purpose of the Study 10
Chapter 2 Materials and Method 12
2.1 Hydrostatic Pressure Experiment 12
2.1.1 Coverslips Surface Modification 12
2.1.2 PA (polyacrylamide) Hydrogels Fabrication 13
2.1.3 Functionalization of PA Hydrogels 15
2.1.4 H9C2 Cell Culture and Hydrostatic Pressure Experiment 17
2.1.5 H9C2 Cells Treated with Rho Inhibitor 17
2.1.6 Immunofluorescence staining 18
2.1.7 Cell Image Capturing and Analyzing 19
2.2 T Cell Cultured in Medium with Different Osmolarity 20
2.2.1 T Cell Preparation 20
2.2.2 Preparation of Medium with Different Osmolarity 20
2.2.3 T Cell Culturing and T Cell Image Analyzing 21
2.2.4 Aquaporin1 (AQP1) Immunofluorescence staining 21
2.3 T Cell Migration in Osmotic Gradient Microfluidic Devices 22
2.3.1 Fabrication of Agarose-based Microfluidic (2D) Device 23
2.3.2 Photolithography of Master Mold for Collagen-filled Microfluidic (3D) Device 25
2.3.3 Soft lithography 26
2.3.4 Preparation of Collagen Solution 27
2.3.5 Integration of Collagen-filled Microfluidic Device 28
2.3.6 Quantification of the Concentration Gradient 29
2.3.7 T Cell Cultured in Collagen-Filled Microfluidics with Osmotic Gradient 29
Chapter 3 Result and Discussion 30
3.1 Fibronectin Density of Different Elastic Hydrogels 30
3.2 H9C2 on Different Elastic Hydrogels under Hydrostatic Pressure 31
3.2.1 Morphological findings of H9C2 Cell 31
3.2.2 H9C2 Cell Area 33
3.2.3 H9C2 Cell Aspect Ratio 34
3.2.4 MyoD and Myogenin Expression 36
3.2.5 Rho Inhibitor Treated H9C2 on 10 kPa Hydrogels 40
3.2.6 Discussion of H9C2 Cell on Different Stiffness Substrates with and without Pressure 41
3.3 T Cell in Medium with Different Osmolarity 42
3.3.1 T Cell Migration in Medium with Different Osmolarity 42
3.3.2 Aquaporin1 Expression of T Cell in Medium with Different Osmolarity 43
3.4 Concentration Generation and Analysis 44
3.4.1 Typan Blue in the Agarose-Based Microfluidics 44
3.4.2 Trypan Blue and Cy3 Gradient in the Collagen-Filled Microfluidics 45
3.5 T Cell Migration in Microfluidic Devices 46
3.5.1 T Cell in the Agarose-Based Microfluidics 46
3.5.2 T Cell Migration in Collagen-Filled Microfluidics 47
Chapter 4 Conclusion and Future Works 49
References 51


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