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研究生:陳昱任
研究生(外文):Yu-JenChen
論文名稱:觀察細胞在利用陽極氧化鋁製造的奈米柱表面上的增殖、黏附、遷移行為。
論文名稱(外文):Cell Proliferation, Adhesion, and Migration on the Nanopillar Srray Structure with Anodic Aluminum Oxide Process.
指導教授:葉明龍葉明龍引用關係
指導教授(外文):Ming-Long Yeh
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物醫學工程學系
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2016
畢業學年度:104
語文別:英文
論文頁數:41
中文關鍵詞:奈米結構表面陽極氧化鋁增殖型態附著爬行
外文關鍵詞:nano structure surfaceAnodic Aluminum Oxideproliferationtopographyadhesionmigration
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在細胞外基質中的周圍自然產生的奈米形態結構已經被證實對於細胞型態、排列、黏著、遷徙、增殖和細胞骨架組織有影響力。在特定的狀態下,微奈米形態對細胞的影響甚至可能取代化學對細胞的刺激。清楚的認識細胞等級的環境所帶來的影響會創造出很多潛在的應用,協助人類在未來生物醫學研究新方向的基礎。因此,研究各種開發的替代方案,用來確定在藉由調查哺乳動物細胞機械傳導線索的相互作來控制細胞功能的重要信號的方式。
在本篇研究中,AAO(陽極氧化鋁)作為模板來複製具有高密度奈米陣列的紫外線固化膠,陣列柱直徑為80奈米和200奈米。培養ADSCs(脂肪幹細胞)在奈米結構表面顯示出紫外線固化膠的生物相容性和物理線索的影響,藉由WST-1細胞增殖實驗。相對於平坦,奈米圖案表面沒有增加細胞的生長速率。此外,電子顯微鏡的圖像顯示奈米柱之間的間隙並不會干擾細胞附著和絲狀偽足的擴展。曠時攝影和傷口癒合實驗分別觀察到ADSCs和MG63(人骨肉瘤細胞)在奈米柱上有著更快的遷徙速度。
免疫螢光染色的結果顯示,ADSCs和MG63在平面組別的粘著斑(focal adhesion)和纖維應力(stress fiber)比其他表面組別具有更好的粘附和更大的貼附面積。曠時攝影的結果顯示,ADSCs在平面的速度(每分鐘0.56微米),在直徑80奈米柱表面為最快(每分鐘1.04微米),直徑200奈米柱表面為次之(每分鐘0.82微米)。特別的地方是,利用此AAO奈米孔洞陣列的基材來探討細胞爬行速度的實驗,根據其結果可得知細胞在有奈米孔洞陣列的結構表面上,其爬行的過程是以個別分散的方式向外爬行,其速度也較快反之在一般培養平面上細胞是以接續原細胞群的移動方式緩慢向外移動。

Within the extracellular matrix, the naturally occurring nanotopographic structures surrounding cells have been proved to have influence on cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization. In a particular case, the influence of micro and nano topographic on cells may even replace chemical stimulation. The clearer recognition of environmental effect at cellular level will create many potential applications and assist human in providing the foundations for new avenues of biomedical research in the near future. Therefore, various studies developed alternative patterns to determine the important signaling modality in controlling cell function by investigating the interaction of mammalian cells with mechanotransductive cues.
In this study, Anodic Aluminum Oxide (AAO) was served as template to replicate UV curable membrane with high density nanopillar array including the diameter of 80 nm and 200 nm. Adipose-derived stem cells (ADSCs) seeded on the nanostructure substrate membranes showed the biocompatibility of UV gel and the effect of physical cues on cell proliferation by WST-1 cell proliferation assay. That is, compared to a flat control, nanopatterned substrates not increased the growth rate of cells. Furthermore, SEM images displayed cell attachment and filopodia extension without interfering by the gap between nanopillars. Time lapse observation and wound healing assay respectively recorded ADSCs and MG63 cells (human osteosarcoma cell line) migrated at a faster speed on the nanopillar surface.
Immunofluorescence showed ADSCs and MG63 have obvious focal adhesion and stress fiber in the control group and have better adhesion and bigger spread area than other groups. By numbers, compared to the speed on the flat control (0.56 µm/min), ADSCs with fastest migration (1.04 µm/min) on the high density nanopillars membrane of 80 nm diameter were markedly higher than the nanopillars membrane of 200 nm diameter (0.82 µm/min). It is noteworthy that MG63 migrated and spread alone on the nanopillars array, which is opposed to the performance of MG63 on the flat substrate that spread slowly with gradual proliferation.

中文摘要 I
Abstract II
致謝 IV
Table of Contents V
List of Tables VI
List of Figures VII
Abbreviation VIII
Chapter 1: Introduction 1
1.1 Nanotechnology in cells research 1
1.2 Fabrication of nanoscale structure 1
1.3 Topographic effect on adipose-derived stem cells 2
1.4 Topographic effect on MG63 cells 3
Chapter 2: Materials and Methods 5
2.1 Flow Chart of Experiment 5
2.2 Reagents and Equipment 5
2.3 Fabrication of Nano-pillars 7
2.4 Adipose-derived stem cells isolation protocol 9
2.5 Culture of Cells 10
2.6 Cell proliferation assays 10
2.7 Scanning Electron Microscopy 11
2.8 Immunocytochemistry 12
2.9 Time-lapse cell migration analysis 12
2.10 Wound healing assay by ibidi 13
Chapter 3: Results and Discussion 15
3.1 AAO surface topography 15
3.2 Stain stem cell markers 18
3.3 Cell proliferation assays 19
3.4 Scanning Electron Microscopy 20
3.5 Immunocytochemistry 22
3.6 Time lapse assay 24
3.7 Wound healing assay by ibidi 26
Chapter 4: Conclusions 28
References 29




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