人類間葉幹細胞及小鼠肌肉母細胞在奈米溝槽生長行為之研究

Abstract

細胞在個體生長過程受到許多外來的訊息所調控，其生長方式會有一定規則排列延伸和移動。本篇論文利用奈米溝槽表面來模擬細胞在細胞所面臨的生長環境，給予方向性指引。以polystyrene（PS）為材質，使用熱壓印的方式在材質表面轉印出寬度300 nm深度為200 nm奈米長型溝槽。分別培養小鼠肌肉母細胞及人類骨髓間葉幹細胞於奈米溝槽表面。觀察細胞在奈米溝槽上生長，進而了解溝槽對於細胞行為之影響。 在小鼠肌肉母細胞方面，本實驗中將小鼠肌肉母細胞培養於奈米溝槽表面，觀察奈米溝槽對於肌肉母細胞融合成肌肉管的影響。結果顯示，肌肉母細胞在溝槽上依順序排列生長。在加入誘導培養基形成肌肉管後則會以平行方式排列於溝槽表面。相較於平面無方向性的肌肉管，此平行肌肉管排列更相似於生物體內肌肉管。此外在溝槽上也可觀察到平行肌肉管會進行側向融合而形成一條更長且多核的肌肉管。 人類間葉幹細胞方面，本研究細胞以BrdU 測定細胞生長速率，並以免疫螢光染色觀察溝槽上細胞骨架。使用骨骼與脂肪之誘導培養基測定溝槽對於間葉性幹細胞分化能力影響。最後再以PCR array 方式測定溝槽對於細胞基因改變。結果顯示，細胞生長為平行排列於溝槽表面，細胞核有拉長現象。此外奈米溝槽會影響間葉幹細胞生長速率，使細胞生長速率緩慢。然而溝槽表面並不會影響細胞分化成骨骼和脂肪的能力。由PCR array的結果分析出和細胞外基質和黏附性有關的基因collage IV α3、integrin α8、MMP13、 MMP14、TMP3 表現有明顯差異。

Cells need signals from the extracelluar matrix to regulate their growth and migration. We designed nanogrooved surface of culture plates to grow cells in specific direction. In this study, we imprinted the polystyrene surface by 300 nm x 400 nm (depth x width). Our purpose is to study the nanogrooved topographical effect on the human mesenchymal stem cells (hMSCs) and the mouse myoblasts (C2C12). First, we studied the pattern of cell growth and myotube formation of C2C12 cells. The result showed that the C2C12 cells were highly aligned along the groove aixs, and the myotubes were also formed in parallel after induction. In contrast to random arrangement on the untreated plates, those parallel arranged myotubes were more mimic the tissue in vivo. Furthermore we found that the parallel arrangment myotubes could fuse laterally. Second parts, we used the BrdU incorporation assay to quantify hMSCs proliferation rate and PCR array to measure the gene expression on the nanogrooved surface. We also treated the cell with osteogenie and adipogenie medium to determine the differentiation ability effect of hMSCs on the nanogrooved surface. The results indicated that the cytoskeleton and nuclei of hMSCs were aligned and elongated along the grooved surface. Cells on the nanogrooaved surface had slower proliferation rate. But there were no significant differences of osteogenesis and adiposgenesis. However, some specific gene expressions of extracellular matrix and adhesion molecules were significant increased including integrin α8 and TMP3 and significant decreased including collage IV α3, MMP13, MMP14.