利用奈米表面調控類神經 PC12 細胞株的生長椎及其樹突型態

Abstract

神經細胞的生長椎決定了神經與神經之間連接的方向，而神經細胞的樹突大量分支 的形成在突觸與突觸之間溝通中扮演重要的角色。奈米尺度表面型態會直接影響細胞的行為。本研究將 PC12 類神經細胞養在 10 nm 至 200 nm 的奈米點陣列上。奈米點陣列結構是藉由陽極氧化鋁製程(AAO)在 TaN 表面的矽晶圓上製備而成。實驗結果發現，未分化的 PC12 細胞在 50 nm 尺度的存活力與 flat 相比成長 169％，在形態方面，未分化 PC12 細胞在 50 nm 尺度上有較多 lamellipodia 觸及到鄰近的細胞且利用 MAP-2 這個蛋白去探討在不同的奈米尺度下未分化 PC12 細胞的形態並無太多差異只有在細胞數目上與存活力的數據呈現一致性。而分化 PC12 細胞在不同的奈米尺度上存活力是沒有明顯差異的。在 50 nm 尺度上生長椎的延展性與 flat 相比增加 448％。在細胞骨架方面，利用 MAP-2 這個蛋白去探討在不同的奈米尺度下，已分化 PC12 細胞樹突型態的不同，我們統計在不同奈米尺度每個細胞的分支點，同樣在 50 nm 上與 flat 相比上升 9 倍。GAP-43 主要表現在樹突及生長椎的位置，藉由其表現在生長椎的螢光強度來印證形態上的結果，發現在 50 nm 的尺度上 GAP-43 的螢光強度與 flat 相比增加 3.9 倍。從上述結果，我們想利用分子的角度去探討奈米表面是如何調控已分化 PC12 細胞的形態差異，所以選用 Wnt 路徑中的 Wnt 7a 和 GSK3 去看這兩個蛋白質的表現量，發現在 50 nm 尺度下，這機制是透過上調 Wnt7a 及下調 GSK3 的蛋白質表現。因此在此次研究我們可以利用特定的奈米尺度去調控已分化 PC12 細胞的生長椎生長，而在樹突形態方面，當神經的分支越多，神經與神經之間的接觸機率也就越大，對於神經訊號傳遞也會產生重大的影響，這對神經再生將有極大的幫助。

We study the effect of topographic nano-cues on neuronal growth-morphology using neuronal-like PC12 cell. The rat adrenal pheochromocytoma PC12 cell is one of the traditional models for the study of growth cone behavior and dendritic morphogenesis. Undifferentiated and differentiated PC12 cell were cultured onto nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by anodic aluminum oxide ( AAO) processing on TaN-coated wafers. Our results revealed that undifferentiated PC12 cell were cultured on nanodot arrays with dot size at 50-nm was significant when compared with on flat control which increased 169%. Undifferentiated PC12 cell seeded on 50 nm nanodot array showed more lamellapodia than the other sizes. On 50 nm nanodot array, it can be seen that lamellapodia can grow to neighboring III

cells. By the cytoskeleton, undifferentiated PC12 cells did not presence neuronal characteristic on nanodot arrays. Differentiated PC12 cell was no significant difference in adherent viable cells between the different size nanodot arrays compared with flat control. Growth cone of differentiated PC12 cells cultured on 50 nm nanodot was significant when compared with on flat control which increased 448%. The results of GAP-43 intensity, which is consistent with the morphology. By the cytoskeleton, differentiated PC12 cells present more dendritic branching on 50 nm nanodot array was significant when compared with on flat control which increased 9 folds. Nanodot arrays regulate up-regulated Wnt7a and down-regulated GSK3 protein expression. Here we show that 50 nm nanodot array control differentiated PC12 cells through Wnt7a pathway. If use in neuronal regeneration, 50 nm nanodot array may cause the axon growth and neuron-neuron connection. In the future, the structure of nanodot array improve the challenges of neural implant and apply on engineering of neuronal regeneration.