巨噬細胞是一種先天免疫細胞，在發炎反應及傷口癒合扮演重要的角色。這些細胞可以極化成促發炎表型(M1)或是抗發炎表型(M2)。先前研究已經證實有許多生物化學因子可以誘發巨噬細胞的極化，但是機械刺激對於巨噬細胞表型的影響目前仍然不清楚。考量不同組織中的微環境硬度有所不同，本篇論文主要研究為探討細胞外物理環境如何影響巨噬細胞表型變化。在這裡我們利用可調節硬度的高分子作為基板模擬不同的組織硬度，進而觀察巨噬細胞在面對不同硬度下的極化情形。另外利用細菌的脂多醣(LPS)和三磷酸腺苷(ATP)誘發巨噬細胞極化成M1表型。我們觀察到隨著硬度的改變，巨噬細胞的形態及遷移模式都有所變化。接著，我們進一步觀察活性氧物質(ROS)的生成及NLRP3發炎小體的形成。我們發現到在較軟的環境下，活性氧物質的增加可能與促進發炎小體的生成相關。另外在基因表現的分析中，巨噬細胞在軟的硬度下，M1表型相關基因表現量較高。綜合實驗所得結果，我們了解到巨噬細胞對於微環境硬度是相當敏感的;當有發炎因子刺激下，在較軟的環境下的巨噬細胞傾向朝向發炎表型極化。我們的研究結果將有助於我們了解組織微環境硬度變化對於巨噬細胞的表型的影響，並且希望可以用來解釋某些疾病的發病機制，提供另一個治療方法開發的方向。

Macrophages can polarize into pro-inflammatory (M1) or anti-inflammatory (M2) phenotypes, which would participate in innate immunity and play an important role in wound healing. Numerous researches indicated that various biochemical signals can trigger the macrophage polarization, but the influences of mechanical stimuli on polarization have not fully explored yet. Considering the different rigidities of different tissues, we investigated the alteration of macrophage phenotypes using rigidity-tunable engineered polymeric substrates. M1 polarization of macrophages was induced by using lipopolysaccharide (LPS) and adenosine triphosphate (ATP), and macrophages showed different morphology and migration pattern in response to the change of substrate stiffness. We further studied the production of mitochondrial reactive oxygen species (ROS) and the formation of NLRP3 inflammasome. Our results revealed that softer substrate facilitate NLRP3 inflammasome activity, which may be associated withup-regulation of ROS production. In addition, higher M1 related gene expression on softer substrate was confirmed with qPCR analysis. In conclusion, these results suggest that the macrophages are mechano-sensitive, and a reducing matrix stiffness can facilitate macrophages to polarize towards an inflammatory phenotype. Our findings are expected to contribute to our understanding of how microenvironment stiffness can influence the macrophage polarization and to provide an alternative mechanotransduction model for macrophage –related diseases.

CONTENTS
中文摘要 I
ABSTRACT II
ACKNOWLEDGEMENT III
CHAPTER 1 1
INTRODUCTION 1
1.1 GENERAL OVERVIEW 1
1.2 STRATEGY AND GOAL OF THE STUDY 3
CHAPTER 2 6
MATERIALS AND METHODS 6
2.1 CELLS AND CELL CULTURE 6
2.2 POLYACRYLAMIDE SUBSTRATE PREPARATION 6
2.3 LPS TREATMENT 8
2.4 CELL SHAPE-DESCRIPTOR ANALYSIS 8
2.5 TIME-LAPSE LIVE CELL IMAGING 8
2.6 MITOSOX ASSAY (OXIDATIVE STRESS ASSAYS) 9
2.7 IMMUNOFLUORESCENCE STAINING 9
2.8 TOTAL RNA ISOLATION 10
2.9 REVERSE TRANSCRIPTION PCR (RT-PCR) 11
2.10 QUANTITATIVE REAL-TIME PCR (QPCR) 11
CHAPTER 3 13
RESULTS 13
3.1 MORPHOLOGY OF J774A.1 CELLS INCUBATED WITH LPS AND ATP. 13
3.2 MORPHOLOGY OF J774A.1 CELLS CULTURE ON POLYACRYLAMIDE GELS WITH VARIOUS STIFFNESS. 13
3.3 EFFECT OF CYTOKINE STIMULATED MACROPHAGE ON CELL MOTION. 16
3.4 CELL MOTION EVOLVED ON VARIOUS STIFFNESS SUBSTRATES. 16
3.5 PRODUCTION OF MITOCHONDRIAL ROS IN STIFFNESS-INDUCED MACROPHAGES. 19
3.6 ACTIVATION OF INFLAMMASOME IN LPS AND ATP-INDUCED MACROPHAGES. 21
3.7 EFFECT OF SUBSTRATE STIFFNESS ON INFLAMMATORY SIGNALING EXPRESSION. 21
3.8 LPS AND ATP TREATMENT INDUCED MACROPHAGES POLARIZATION STATE. 25
3.9 EFFECT OF STIFFNESS ON MACROPHAGES POLARIZATION STATE. 25
CHAPTER 4 28
DISCUSSION 28
4.1 CELL SHAPE WAS ASSOCIATED WITH MACROPHAGE POLARIZATION. 28
4.2 CELL MOTION DIFFERENCE BETWEEN LPS AND ATP-INDUCED AND STIFFNESS-INDUCED MACROPHAGES. 28
4.3 MITOCHONDRIAL ROS PRODUCTION IN MACROPHAGE POLARIZATION. 29
4.4 THE SOFTER STIFFNESS PROMOTED ACTIVATION OF THE NLRP3 INFLAMMASOME. 29
4.5 EFFECT OF STIFFNESS ON MACROPHAGE PHENOTYPE POLARIZATION. 30
4.6 SUMMARY 30
CHAPTER 5 32
REFERENCES 32

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