臺灣博碩士論文加值系統

　　本實驗以473nm藍光雷射作為刺激，利用雷射形成微圖樣的方式，來誘導癌細胞進行遷移。藉由投射不同函數梯度之微圖樣，觀察對癌細胞遷移行為之影響，並探討活性氧類分子 (reactive oxygen species, ROS)在此過程中扮演何種角色。
　　本研究使用空間光調變器 (spatial light modulator)來調控藍光雷射，使雷射光束的橫切面能形成任意的微圖樣，並且成像於顯微鏡的載物台之上。接著以線性函數、二次函數以及四次函數三種不同光強度梯度的微圖樣來照射癌細胞，並比較癌細胞在不同的照射條件下，其X軸方向的位移量的差異，藉此探討不同的光梯度對癌細胞遷移造成之影響。本實驗發現癌細胞在四次函數光梯度微圖樣的照射下，癌細胞的遷移距離最遠，因此推論A549細胞在此光梯度照射下，細胞遷移效果最佳。
　　接下來，進一步探討ROS在此過程中扮演的角色，本實驗以螢光染劑 (CM-H2DCFDA)標定細胞內的ROS，藉此觀察到A549癌細胞體內的ROS含量與藍光光強度值，呈現正相關的特性。以藍光形成的光強度微圖樣，能使癌細胞朝著特定方向進行遷移行為。與此相反的是，在培養液含有抗氧化劑的環境裡，此效果被大幅度地降低。然而，人類肺纖維母細胞 (MRC-5)在低藍光光強度的照射下，其體內的ROS已達到飽和狀態。因此MRC-5細胞在藍光光強度微圖樣的刺激下，並沒有朝著特定的方向進行遷移行為。

We project different optical micro-pattern of a 473 nm blue light laser with various intensity gradients on a single lung cancer cell. We investigate the role of reactive oxygen species (ROS) in the mechanism of blue light induced cell migration behavior.
A spatial light modulator (SLM) is used to generate 473 nm laser pattern imaged onto the focal plane of a microscope objective. We use a linear, a quadratic and a quartic optical intensity gradient to irradiate an individual lung cancer cell (A549). To compare with the cancer cell migration under different illumination conditions, we measure the cell displacement along X-axis direction. The A549 cell exhibited the most obvious directional migration away from the gradient of a quartic optical intensity gradient.
We observed that the intracellular amounts of ROS were proportional to the intensity of the blue light, and the blue light intensity gradient could drive directional A549 cell migration. This optically induced directional A549 cell migration was inhibited by a ROS scavenger (Trolox) in the culture medium in a dose-dependent manner. In contrast, the ROS levels in fibroblasts were saturated by the blue light at low intensity and therefore the fibroblast (MRC-5) did not exhibit a directional migration in the intensity gradient pattern.

致謝 i
摘要 iv
Abstract v
目錄 vi
圖目錄 viii
第一章 研究背景與動機 1
1.1癌細胞的轉移 1
1.2細胞遷移 2
1.3群體細胞遷移 4
1.4光與細胞之間的交互作用 5
1.4.1以雷射光點刺激板狀偽足調控細胞的遷移方向 5
1.4.2以微圖樣限制細胞生長區域 7
1.4.3以動態的微圖樣調控細胞遷移 10
1.5藍光對細胞的影響情形 11
1.5.1活性氧類與藍光之間的關聯性 11
1.6 研究動機 16
第二章 實驗系統 17
2.1光學系統 17
2.2系統架設的方法 21
2.3儀器規格與參數 25
第三章 實驗方法 28
3.1實驗設計 28
3.1.1 細胞內ROS的含量與光強度之關係 28
3.1.2 光梯度圖形刺激細胞進行遷移 29
3.2材料與方法 31
3.2.1 細胞株培養 31
3.2.2 ROS的螢光標定 31
3.2.3 調配抗氧化劑的濃度與使用 32
3.2.4 染色的步驟與注意事項 33
3.2.5 螢光亮度的量化方法 33
3.2.6 雷射光照射周期與自動化拍攝系統 34
3.2.7 記錄細胞遷移軌跡的方法 35
第四章 結果與討論 37
4.1比較藍光照射前後細胞體內ROS的差異 37
4.1.1 藍光與MRC-5體內ROS之關係 37
4.1.2 比較A549細胞加入Trolox前後的差異 38
4.2以藍光形成的光強度梯度誘發細胞遷移 40
4.3減少活性氧類的產生對細胞遷移的影響 48
4.4 結論 52
第五章 未來展望 53
參考文獻 55

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