次微米溝脊表面對細胞貼附及功能之影響

Modulation o fthe Adhesion and Function of Cells on Submicro-Grooved Surfaces

林佳樺(Jia-Hua Lin)

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(2008年)

https://doi.org/10.6342/NTU.2008.02459

摘要

細胞在生理環境中接觸不同尺寸的奈微米表面，同時受到表面物理性質的刺激。因此本研究利用具有不同次微米、奈米尺寸的矽晶片或聚苯乙烯（PS）表面（100∼500 nm不同溝寬或100∼400 nm不同溝深）應用在肝瘤母細胞及初級新生大鼠心肌細胞上，經過特定的培養時間後，觀察並分析細胞的型態及功能。期望透過特定的地形表面改變細胞型態，進而觀察細胞受到物理刺激後功能之影響。
首先，將肝瘤母細胞佈植在不同脊寬的直溝矽晶片地形表面（100、200、300、400、500 nm），並觀察培養一天之後細胞的行為表面。結果當脊寬小於300 nm時，細胞型態隨著脊寬的增加而明顯改變，同時，細胞的貼附情形亦隨著脊寬的減少而降低，但是肝瘤母細胞仍然能夠反應100 nm的地形表面。當脊寬介於300∼500 nm之間時，脊寬的改變對細胞型態的影響並沒有很明顯，但是細胞仍然能夠受到地形導引而改變。另外，肝瘤母細胞在不同溝深的矽晶片表面（110、180、380 nm）上，細胞攤開及拉伸的情況皆受到明顯的影響，隨著溝深加深而增加。此外，透過雷射共軛焦螢光顯微鏡的觀察，細胞在有溝脊的區域上，隨著溝深的增加，不僅較快形成細胞骨架，同時白蛋白（albumin）的分泌情形也比細胞在平坦表面上面來得多。
接著在矽晶片表面塗佈上矽鏈薄層，轉印出具彈性、可重複使用的二甲基矽氧烷（PDMS）模具，二次轉印出類似矽晶片表面的PS溝脊表面。除了細胞型態的分析之外，也分析肝細胞特有的功能表現，白蛋白分泌及尿素轉換能力。肝細胞在特定圖案地形表面上，有較高的白蛋白分泌量，但是尿素的轉換能力隨著溝深的加深而減低。整體而言，細胞受到表面地形物理刺激的些微拉伸，能夠刺激白蛋白的增加，同時亦能夠維持其尿素轉換的解毒能力。
另外，將初級新生大鼠心肌細胞培養在具有特定地形的PS表面上，佈植低細胞密度以觀察細胞對表面之行為；佈植高細胞密度觀察細胞與細胞間的關係。在低細胞密度時，細胞在深溝的表面（350 nm）隨著溝深加深而伸展，同時能夠良好地控制細胞延伸的方向；當佈植高細胞密度時，隨著細胞移動性的增加，細胞能夠貼附、融合，進而形成較接近成熟大鼠心臟組織的結構。因此，表面的地形修飾能夠幫助導引心肌細胞的排列，期望能夠對心臟組織工程的發展有所貢獻。

關鍵字

次微米表面 ； 肝細胞 ； 白蛋白 ； 尿素 ； 心肌細胞

並列摘要

Cells are well known to respond to topographic cues in their natural nanometer-scale environment. In this study, the pattern-designed silicon or PS surfaces with various geometries (100 to 500 nm in widths or 100 to 400 nm in depths) would be applied to the hepatoblastoma cell, HepG2/C3A cells, or the primary rat cardiac muscle cells for specific time and analyzed. Furthermore, the relationship between the varied topography dimensions and the cellular behaviors would be explored when the cells were affected by the surface guidance.
First, the patterned silicon surfaces with different ridge widths (from 100, 200, 300, 400, to 500 nm, and 350 nm in depth) would be seeded with C3A cells for 1 day. The cell morphology showed that cell spreading area and elongation increased with increasing ridge widths when the ridge width was smaller than 300 nm. However, there was no significant difference between the cells on 300- to 500-nm-deep grooved surfaces, but cells would be guided for all scale patterned surfaces as well. Then, the grooved silicon surfaces with various groove depths (110, 180, and 380 nm) were cultured with C3A cells and analyzed. The results showed that the cell spreading and elongation increased with increasing groove depths. Besides, both of the F-actin fibers and the albumin secretion in the cells on the grooved surfaces were observed earlier than those on the flat region.
In addition, the imprinted PS surfaces with various depths were replicated by PDMS molds which were cast from the silicon masters. Besides the morphology characteristic analysis, both of the albumin secretion and urea conversion ability from the C3A cells were determined. The albumin expression was improved by the patterned surfaces but decreased with increasing groove depths. Furthermore, the urea conversion ability was reduced with increasing depths. In general, the slight stretch, due to the topographic effect, would enhance the albumin secretion and maintain the urea conversion ability.
For the cardiac muscle cells on the designed topographic PS surfaces, the cell-surface interactions were examined under a low seeding cell density. The results showed that the degree of cell alignment of the cells on the deep-grooved surfaces (350 nm) was the best. Moreover, cells were seeded under a high cell density for the cell-cell interaction observation. The oriented cells on the patterned surfaces connected to each other, and the architecture of the cells were similar to the structure in a native rat heart tissue. Hence, the morphology of the primary cardiomyocytes would be controlled by the surface topography and formed the structure similar to a native heart tissue. The results may be interesting for developing the heart tissue regeneration.

並列關鍵字

micropatterned surface ； HepG2/C3A ； albumin ； urea ； cardiomyocytes

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