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系統識別號 U0026-2708201011261600
論文名稱(中文) 利用生物微機電製程及方向性流體剪力探討細胞黏附力和黏附相關蛋白之力學反應
論文名稱(英文) Investigating integrin-FAK-Src intracellular signaling in response to micropatterned geometry and directional shear stress
校院名稱 成功大學
系所名稱(中) 細胞生物及解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 98
學期 2
出版年 99
研究生(中文) 黃惠君
研究生(英文) Hui-Chun Huang
學號 T9697102
學位類別 碩士
語文別 英文
論文頁數 65頁
口試委員 指導教授-吳佳慶
口試委員-黃步敏
口試委員-湯銘哲
口試委員-蘇芳慶
中文關鍵字 FAK  Src  生物微機電製程  細胞黏附力  流體剪力 
英文關鍵字 FAK  Src  Bio-MEMS  micropatterning  cell adhesion force  shear stress 
學科別分類
中文摘要 心血管系統對於人體代謝和生理上扮演著重要角色,循環系統的主要功能是體內運送血液的器官和組織,主要包括心臟、血管。在物理力學上,與血管系統有密切的相關性就是流體動力學,血管內皮細胞是最主要受到流體力學影響,此機械力含有血流剪力(LSS)以及微環境,這些因子可以調控細胞的生理平衡、增生、凋亡以及功能表現。
先前研究指示出利用生物微機電製程,模擬內皮細胞在微環境當中,發現當內皮細胞受到異相性幾何力限制可引起細胞凋亡,若施與平行於內皮細胞所生長方向之流體剪力則可增加細胞存活率。細胞是如何感受到外來刺激進而影響細胞內蛋白與蛋白之間訊息傳遞仍然未知。已知細胞膜附近的黏著蛋白酶 (FAK) 會透過 Integrin 與細胞外基質(ECM) 接觸並形成黏附點,而磷酸化之 FAK 可活化黏著相關分子 (Src),並啟動一聯串之細胞內訊息傳遞。本研究將內皮細胞生長在不同寬度的微環境幾何圖形中以及血流剪力做為力學應用的條件刺激,並探討此刺激對於內皮細胞integrin-FAK-Src的細胞內訊息傳遞。由實驗結果發現LSS引起動脈內皮細胞形態的方向性平行於剪力方向,然而靜脈內皮細胞則垂直於LSS。當給予不同方向性之外力時,平行和垂直的LSS會誘導動脈內皮細胞的pFAK分布於血流剪力下游。在靜脈中,卻只有垂直方向性LSS影響pFAK分佈於血流剪力下游。我們並利用免疫染色觀察pSrc表現,當多細胞培養的影像顯示出當動脈內皮細胞受到LSS刺激使的pSrc主要位在細胞邊緣,而在靜脈中則沒有顯著變化。在不同方向性流體刺激時,平行的LSS會使動脈內皮細胞減少pSrc強度;相對地,在靜脈中則沒有變化。若考慮FAK和Src活化分布會有空間與時間上的影響,因此,我們利用GFP-FAK和FRET-Src biosensor來觀察活體細胞內蛋白表現,當動脈內皮細胞受到不同方向性LSS會促使FAK有更快速的更新率(turnover rate)。FRET-membrane tagged Src則在動脈內皮預攤付周圍以及受異相性幾何力限制的細胞膜呈現不活化狀態。LSS也會刺激Src在動脈內皮下游中不活化。對於內皮細胞而言,當細胞處於外在機械力的刺激下,細胞受到刺激(Mechanicalsensing)進而轉換成細胞內的訊息傳遞因子稱為力學訊息傳遞(Mechanotransduction)。透過本研究可以利用微奈米技術了解細胞以及組織去模擬真實人體微環境,並探討訊息傳遞的重要性,進而修復已受損或者缺陷的組織以及器官,達到組織工程以及再生醫學的目的。
英文摘要 The mechanical environment and shear stress play essential role in cardiovascular system for endothelial cell (ECs) homeostasis, proliferation, apoptosis, and functional expression. How the cell senses mechanical stimulation is still poorly understood in cellular biomechanics. In focal adhesions (FAs), focal adhesion kinase (FAK) binds to integrin for cell attaching to the extracellular matrix (ECM). Phosphorylation of FAK (p-FAK) activates Src and initiates intracellular signal cascade for signal transduction. The purpose of this study is to investigate the effect by anisotropic cell morphology and directionality of mechanical force (shear stress) on FAs of ECs. The bovine aortic endothelial cells (BAECs) and bovine vena cava endothelial cells (BVECs) were constrained by culturing on micropatterned (MP) strips using BioMEMS lithographic technology. Cells on 30 and 60 μm MP strips showed the p-FAK was evenly distributed in cytosol and formed focal adhesion. On 15 μm MP strips, cells occurred elongation morphology, p-FAK localized at peripheral of cell membrane and linked with the end of stress fibers. After cells growing for 24 hr, the cells were subjected to laminar shear stress (LSS, 12 dyne/cm2) in parallel or perpendicular direction for 24 hr. Without MP, LSS induced parallel cell alignment with enhanced p-FAK at the downstream of LSS direction in BAECs, while perpendicular orientation with flow direction was observed in BVECs. The p-Src appeared at the cell border in monolayer of BAECs under both static and flow condition. The expression of p-Src was not significant distributed at the border under static and flow condition in BVECs. Applying the directionality of LSS, BAECs had lower pSrc expression under parallel flow, but not in perpendicular flow. In contrast, the expression of pY416Src was not significantly difference in BVECs under both static and directionality of LSS condition. Next, we visualized the molecular dynamics of FAK by tagging FAK with green fluorescence protein (GFP) to display the cell-substrate interaction in various microenvironments. By fluorescent resonance energy transfer (FRET) system, the CFP/YFP-tagged Src plasmid was also established to observe the activation of Src in living cells. Shear stress-induced p-FAK and FRET-Src activation is rapid at the downstream of flow direction in BAECs. The increase of FAK turnover rate was confirmed by accelerating the FAK assembling and dissembling as the BAEC subjected to LSS. These results suggesting the mechanical environment and shear stress affect the different responses of ECs. These results in arterial and venous ECs might explain the pathophysiological mechanism of restenosis in vein graft. The biomechanical information obtained from endothelial model may have clinical relevance in creating the microenvironment for tissue repairing or stem cell differentiation to achieve the tissue engineering and regenerative medicine.
論文目次 中文摘要 5
ABSTRACT 7
INTRODUCTION 9
SHEAR STRESS RESPONSES TO ENDOTHELIAL CELL 9
CELL ADHESION AND MECHANOTRANSDUCTION 11
MOLECULAR SIGNALING OF FAK AND SRC 12
DIFFERENCE BETWEEN ARTERY AND VEIN 15
SUBSTRATE GEOMETRY AND BIO-MICRO-ELECTRO-MECHANICAL SYSTEM (BIO-MEMS) 15
SPECIFIC AIMS 18
MATERIAL AND METHODS 20
ENDOTHELIAL CELL CULTURE 20
MICROPATTERNED ECM MICROENVIRONMENT 20
PLASMID TRANSFECTION FOR LIVING CELL IMAGES 21
LIVING CELL GFP AND FRET MICROSCOPIC SYSTEM WITH MICROPATTERNED ECM ENVIRONMENT 22
IMMUNOFLUORESECNT STAINING 22
SHEAR STRESS EXPERIMENTS 23
RESULTS 25
SHEAR STRESS CAUSED THE DIFFERENT RESPONSE OF FAK DISTRIBUTION AND SRC ACTIVATION IN THE ECS OF ARTERY AND VEIN 25
THE SPATIAL CHARACTERISTIC OF PFAK AND SRC ACTIVATION ON MICROPATTERNED ECM 31
DISTRIBUTION OF FAK PHOSPHORYLATION BY DIRECTIONALITY OF SHEAR STRESS IN A/V ECS 34
ESTABLISHMENT OF LIVING CELL GFP-FAK SYSTEM FOR MEASURING THE TEMPORAL-SPATIAL DISTRIBUTION OF PFAK 40
THE TEMPORAL-SPATIAL DISTRIBUTION OF PFAK AND SRC DYNAMICS IN LIVING CELL ON MP SURFACE AND SUBJECTED TO SHEAR STRESS WITH DIFFERENT DIRECTIONALITIES 42
ESTABLISHMENT OF LIVING CELL FRET-SRC SYSTEM FOR MEASURING THE TEMPORAL-SPATIAL DISTRIBUTION OF SRC ACTIVITIES 47
THE TEMPORAL-SPATIAL CHARARISTICS OF SRC ACTIVATION IN LIVING CELL ON MP SURFACE AND SUBJECTED TO SHEAR STRESS WITH DIFFERENT DIRECTIONALITIES 49
DISCUSSION 53
CONCLUSIONS 57
REFERENCE 59

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