利用飛秒雷射技術及順向電紡織方法 分別建構二維和三維結構並應用於神經細胞PC12特性之探討

Abstract

由於神經細胞網路之間的溝通在神經科學領域始終是個謎團，我們希望藉由建構特定結構誘導PC12神經細胞生長成特定網路以利神經網路的探討。因此，在本篇論文中我們分別利用飛秒雷射系統建構二維結構以及順向電紡織方法建構三維結構。在二維結構的建構上，在玻璃基材表面塗覆上具有斥細胞性質的聚合層(MPC polymer) 後利用特定能量的飛秒雷射燒蝕使玻璃表面裸露，再利用poly-L-Lysine塗覆而將原本裸露的玻璃表面轉變為親細胞性。在三維結構的建構上，我們採用生物相容性材料，聚乳酸（PLA）作為基材，利用電紡織技術結合鋁片簍空收集器及外加電場的控制，使聚乳酸可順向排列於收集器上，而後利用電漿技術及poly-L-Lysine塗覆增加其細胞貼附性。結構建構完成後將PC12培養於結構上模擬細胞網路，並藉由螢光染色觀察PC12 軸突和樹突之生長情形。結果顯示二維結構可使細胞貼附於特定位置上甚至能誘導PC12軸突和樹突的生長方向，但因為雷射聚焦的限制使得此方法成功機率極低，而三維結構則是能限制細胞於一維方向生長並在細胞間形成連結可做後續研究。因此我們利用實驗室先前所合成出來的FITC-dopamine來觀察PC12 細胞網路間的溝通，可惜的是因為此3D結構懸浮於溶液中，共軛焦顯微鏡無法來觀察其FITC-dopamine的釋放必須再做固定化或其他的方式改良以利實驗進行。

The communication in the neuron network is still a mystery. We expect to fabricate specific scaffold to induce PC12 growth so that we can easily survey the characteristics of neuron network. Herein, we fabricate 2D scaffold by femtosecond laser system and 3D scaffold by aligned electrospinning method. In 2D scaffold fabrication, we used glass substrate coated by cytophobic MPC polymer and utilized femto second laser system to ablate the MPC polymer in specific part. Then, poly-L-Lysine coating change the cytophilicity of glass surface. In 3D scaffold fabrication, we chose a kind of biocompatible material PLA as our model. Combining electrospinning with aluminium hollow collector and applied electron field, we can get the aligned PLA on the aluminium hollw. Then, fluorescence stain was used to observe the PC12 growth. The result showed that PC12 can well-attached on the 2D scaffold even controlled the growth direction by the 2D scaffold. Because of the laser focus limitation, we cannot improve their quality. In contrast, 3D scaffold can restrict PC12 to grow in one direction and connect to each other so that we can do the further experiment by this method. Finally, we used FITC-dopamine to observe the communication between PC12. Due to the floating scaffold, it is difficult to observe the FITC-dopamine release from cells in right focus. We need to immobilize the scaffold to assist the observation by confocal microscopy.