臺灣博碩士論文加值系統

本研究是將人類臍靜脈內皮細胞(Human Umbilical Vein Endothelial Cell Line, ECV304)培養在塗佈有膠原蛋白及纖維網蛋白的生醫材料聚二甲基矽氧烷(Polydimethylsiloxane, PDMS)上，應用介電泳力來量測內皮細胞貼附能力，以電壓驅動電極產生之介電泳力表示細胞貼附能力，並加入蛋白質檢測技術，觀察細胞貼附能力與蛋白質活化關係。比較當細胞培養於塗佈有膠原蛋白及纖維網蛋白的PDMS上，貼附能力與蛋白質活化是否有差異，並比較膠原蛋白與纖維網蛋白當中哪一種細胞外基質(Extracellular Matrix, ECM)能提供細胞一個較穩定的生長環境，而更適合運用於組織工程方面。
由實驗結果顯示，當細胞培養於塗佈有膠原蛋白的PDMS上其貼附能力較不穩定，於第二及第五小時有貼附能力增加趨勢，且焦點黏附激&;#37238;(Focal Adhesion Kinase, FAK)於第二及第五小時有活化的現象；而當細胞培養於塗佈有纖維網蛋白的PDMS上其貼附能力較為穩定，其貼附能力隨著時間有明顯遞增，且FAK活化現象隨著時間有增加趨勢。就蛋白質活化來說，由初步實驗發現FAK活化與細胞貼附能力是有高度相關性。
本研究嘗試加入AG18、Genistein及LY294002這三種抑制劑來抑制蛋白質酪胺酸磷酸化，發現Genistein在抑制蛋白活化及貼附能力方面效果較好，然而綜觀所有實驗可發現FAK在細胞貼附能力中扮演著一重要的角色。

This study investigated the relationship between the cell adhesion and activation of protein when the human umbilical vein endothelial cells (Human Umbilical Vein Endothelial Cell Line, ECV304) cultured in a coating of collagen protein and fibronectin of biomedical materials-dimethyl siloxane (Polydimethylsiloxane, PDMS). The electrophoresis force is used to measure the attached ability of ECV304 then driving voltage of electrodes to produce the electrophoresis force meant the adhesive ability of cell. The difference between the adhesive ability of cell and activation of protein is compared under ECV304 cells cultured in a coating of collagen protein and fibronectin on PDMS. Then find out which extracellular matrix (Extracellular Matrix, ECM) cells can provide a more stable growth environment for cell for the collagen protein and fibronectin.
The results revealed that when cells cultured in a collagen protein coating on the PDMS which adhesive ability is more instability. The adhesive ability for the second and fifth hours increased and the activation of the focal adhesion kinase (Focal Adhesion Kinase, FAK) also increased for the second and fifth hours. When cells cultured in a coating of the fibronectin attached PDMS which adhesive ability is more stable. It showed that adhesive ability increased as cultured time and FAK activation phenomenon also increased as time. On the activation of protein, the experiment showed the significant relationship between the adhesive ability of cell and FAK activation phenomenon.

This study attempts to join three inhibitors, AG18, Genistein and LY294002 to curb Lao Ansuan protein phosphorylation. It shows Genistein on the adhesive ability and the inhibition of protein activation is more effective. However FAK may be an important role on the adhesive ability of cell.

摘要 i
Abstract ii
符號說明 iv
目錄 vi
圖表目錄 i
第一章 緒論 1
1.1 微機電系統 2
1.2 細胞黏附機制 3
1.3 細胞訊息傳遞機制 6
1.4 研究動機 8
1.5 研究目的 9
第二章 理論與數值模擬 10
2.1 膠原蛋白(Collagen)與纖維網蛋白(Fibronectin) 10
2.2 蛋白質磷酸化(Phosphorylation) 11
2.3焦點黏附激&;#37238;(Focal Adhesion Kinase, FAK) 13
2.4 介電泳理論 13
2.5 數值模擬 21
第三章 實驗設備與方法 24
3.1 實驗設備 25
3.2 實驗材料、製作與方法 26
3.3 PDMS基板之澆鑄 29
3.4 鋁電極板製作流程 30
3.5 PDMS 基板前處理 33
3.6內皮細胞前處理 34
3.7 西方點墨法(Western Blot Analysis) 35
3.8 細胞貼附能力量測 39
3.9 抑制劑分析 42
第四章 結果與討論 43
4.1 西方點墨法分析結果 43
4.2 貼附能力量測 47
4.3加抑制劑於特定時間點之西方點墨法分析 50
4.4 加抑制劑於特定時間點之貼附能力比較 54
第五章 結論與建議 61
5.1結論 61
5.2 建議 65
參考文獻 66

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