次微米溝脊地形與表面化學性質對於神經細胞分化之影響

Influence of Submicron-Grooved Topography and Chemical Surface Properties on Differentiation of Neural Cells

陳政宏(Cheng-Hung Chen)

指導教授 ： 蔡偉博

國立臺灣大學/工學院/化學工程學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201603497

摘要

次微米溝脊表面和生物活性因子可以提供物理化學線索控制神經細胞的增生與分化，幹細胞具有自我再生與分化成各種細胞譜系的能力包含神經細胞，而PC12細胞經過神經生長因子(NGF)刺激後容易分化成神經細胞，這使得PC12細胞被廣泛應用於神經分化的研究。次微米溝脊表面改質可以促進細胞貼附與增生，過去許多研究顯示聚多巴胺(Poly-dopamine)處理過的表面能夠促進細胞貼附。在本實驗中，我們利用聚多巴胺修飾不同寬度(400與800奈米)與不同深度(100與400奈米)的聚苯乙烯(PS)次微米溝脊表面，並將PC12細胞、人類脂肪幹細胞(hADSCs)與人類臍帶血幹細胞(UCB-MSCs)培養於次微米溝脊表面，利用含有神經誘導生長因子的培養基中進行神經分化，觀察並分析細胞突觸型態，接著使用Tuj-1(神經元標記物)、GFAP(星形膠質細胞標記物)免疫染色，比較不同神經細胞在不同表面神經分化的程度。
我們的研究結果顯示，PC12細胞培養在平坦表面有多個突觸產生，而培養在溝脊表面則主要產生兩個突觸且沿著溝槽方向排列。此外結果也顯示，地形深度能夠促進突觸的生長與排列，因此400/400奈米與800/400奈米(寬度/深度)的次微米溝脊表面能夠提升PC12細胞、hADSCs、UCB-MSCs的突觸形成比例、突觸長度與Tuj-1表現比例。我們的研究結果顯示次微米溝脊表面和神經生長因子能夠促進突觸生長並且誘導細胞分化成神經細胞。

關鍵字

神經分化 ； 次微米地形 ； 幹細胞 ； 表面改質

並列摘要

Surface topography and bioactive molecules can generate physicochemical cues that control proliferation and differentiation of neural cells. Stem cells have ability to self-renew and differentiate into various cell lineages including neuronal cells. Pheochromocytoma 12 (PC12) cells are prone to differentiate into neuron-like cells with nerve growth factor (NGF) treatment which makes PC12 cells are a good model for neuronal differentiation research. Surface modification of submicron-grooved topography is requiring for improving cell adhesion and proliferation. Our previous study indicated that poly-dopamine (PDA) coated surface can promote cell adhesion due to an enhancement of immobilized serum adhesive proteins and adhesion peptides onto the substrates.
In this study, PDA was used to modify polystyrene (PS) submicron-patterns with different widths (400 and 800 nm) and depths (100 and 400 nm). We examined neurites of PC12 cells, human adipose-derived stem cells (hADSCs) and human umbilical cord blood-derived MSCs (UCB-MSCs) which were incubated in neuronal induction medium containing growth factors. Then, the differentiated cells on different grooved topographies were immunologically stained by Tuj-1 (a neuron marker) and GFAP (an astrocyte marker) to compare the extent of neuronal differentiation.
Our results illustrated that PC12 cells on grooved topography have predominantly bipolar neurites which align along the direction of the patterns while flat surface have multiple neurites. We can conclude that the depths of topography have strong impact on neurites outgrowth and alignment. The 400/400 and 800/400 nm (widths/depths) PS grooves are appropriate for PC12 cells, hADSCs and UCB-MSCs to enhance percentage of neurites, neurites length and percentage of Tuj-1 positive cells. Therefore, our data suggest that submicron-grooved topography and neurotrophic growth factors supported neurites outgrown and differentiated into neuron-like cells.

並列關鍵字

neuronal differentiation ； submicron-grooved topography ； stem cells ； PC12 cells ； surface modification ； poly-dopamine

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