由於奈米技術迅速的發展，我們生活中充斥著大量的奈米產品，像是奈米保養品、防曬用品、奈米銀抗菌產品等，而奈米粒子多會經由呼吸道吸入與皮膚殘留兩種途徑進入體內，進入體內後再通過細胞膜上孔洞進入細胞內，影響細胞的生理功能，產生毒性。研究指出奈米粒子的累積不僅會造成細胞傷害，累積在肝臟腎臟等器官中，更會進一步造成多種退化性疾病、老化、突變以及癌症的形成。
　　為評估奈米粒子對生物造成的傷害，我們發展一套能提高偵測極限的生物感測方法，利用微接觸壓印技術製作出微米等級的圖案化細胞，藉由細胞型態的改變，測試不同藥物於不同品系及形狀差異間的細胞產生毒性的情形。結果發現被限制於特定形狀的細胞能提升偵測極限，比對照組更為敏銳。在二氧化鈦上，低於文獻指出產生毒性濃度的1/10便可被偵測到藥物的存在，成功地達到本實驗設計的目的。
　　我們利用螢光漂白恢復技術，探討偵測極限提高的可能細胞機制。利用微接觸壓印的膠原蛋白圖案作為細胞外基質成分，觀察細胞內肌動蛋白細胞骨架動態會隨細胞外基質間的交互作用而改變。考量細胞骨架動態與胞內能量代謝相關，推測肌動蛋白的調節會與前一項實驗中ROS表達量有關。
綜合上述陳述，透過微接觸壓印技術產生的微米級圖案化細胞，不僅讓我們發展出高通量的毒性測試生物方法，並且提升偵測極限與靈敏度，可大幅度節省醫學檢測上珍貴的檢體。其中透過觀察不同型態的細胞之細胞骨架的排列情形與動態調節功能，可作為研究細胞生理功能及與細胞外基質間交互作用的模型。

With the rapid growth of nanotechnology development, numerous products contained nanoparticles are widely used in our daily life. However, the nanoparticle can accumulate in our bodies via exposure of nanoparticle in environment . The accumulation of nanoparticles can cause cell damage, degenerative diseases, and even cancer progression. Thus, there is an increasing concern about the nanoparticle utilization recently, and an emerging need for nanotoxicity detection.
In this study, we aimed to develop a cell-based nanotoxicity biosensor with high detection sensitivity. Micro contact printing (mCP) was applied to pattern liver cells into specific shape, and the nanotoxicity of nanoparticle was quantified with fluorescent ROS indicator using fluorescent microscopy. Our results demonstrated, comparing to control group, the patterned cells have higher sensitivity to nanoparticles. Under the pressure of TiO2, our designed cell can detect (0.625 mg/L) TiO2 presented in the medium.
We further investigated the underlying mechanism of sensitivity enhancement. With FRAP microscopy, decreasing actin turnover of pattern cells was observed, and the slower actin dynamics is associated with ROS expression. In addition, considering the spatial organization of mitochondria within patterned cells, the mitochondrial RIRR model may contribute to the ROS generation.
In summary, we demostrated that the micron-scale patterned cells fabricated by mCP, can be ultilized as a more effective nano-toxicity biosensor, and observed the rearrangement of cytoskeleton and actin dynamic in cells are highly associated with cell physiological functions.

摘要 ii
Abstract iii
目錄 iv
圖表目錄 vi
Chapter 1 文獻回顧 1
1.1 奈米毒性 (Nanotoxicity) 1
1.2 奈米毒性的作用機制 2
1.3 細胞型態生物感測器 3
1.4 細胞模型 6
1.5 微接觸壓印(Microcontact Printing, μCP) 6
1.6 光漂白螢光恢復系統( Fluorescence recovery after photobleaching，FRAP) 10
1.7 細胞代謝與肌動蛋白的關係 11
Chapter 2 材料與方法 13
2.1 細胞培養 13
2.2 圖案化細胞的實驗流程 13
2.3 製作印章 14
2.4 聚苯乙烯玻片的製備 18
2.5微接觸壓印 ( Microcontact printing，μCP) 19
2.6圖案化細胞 20
2.7免疫螢光染色法 20
2.8 細胞層級的生物傳感器－活性氧物質試驗 21
2.9光漂白螢光恢復顯微系統的建立 22
2.10 資料統計整理方法 23
2.11 實驗材料與儀器 23
Chapter 3 結果與討論 25
3.1於聚苯乙烯玻片上壓印的微小圖案 25
3.2 於基材上培養出微米級圖案化細胞 27
3.2.1 由免疫螢光染色觀察細胞之細胞骨架排列 27
3.2.2 量化免疫螢光染色之分析 31
3.3 將圖案化細胞應用於奈米毒性藥物檢測 37
3.3.1 使用標準品t-BHP對肝細胞產生毒性 37
3.3.2 使用奈米微粒二氧化鈦(TiO2)對肝細胞產生奈米毒性 44
3.4 由光漂白螢光恢復 (FRAP)顯微系統量測肌動蛋白動力學，並揭示其與活性氧物質產生之關係 50
Chapter 4 結論 55
參考文獻 57

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