以奈米點陣列操控肺腫瘤細胞纖維蛋白溶解、細胞貼附和存活能力

Abstract

材料在生物工程的應用上扮演重要的角色。了評估奈米表面對細胞的影響，本研究將小鼠肺腫瘤細胞培養在10至200奈米點陣列上，奈米點陣列結構是藉由陽極氧化鋁製程在鉭化氮表面的矽晶圓上製備而成。奈米點陣列的大小對細胞的生長、增生、附著、骨架形成和基因表現是我們主要研究重點。 經由實驗結果發現，當表面細胞密度達到飽和，小鼠肺腫瘤細胞在五十奈米的點陣列上仍有最佳的存活率，細胞培養七十二小時在100和200奈米的點陣列上數量減少了34.7%和46.71%。細胞培養在50奈米點表面有最大的貼附面積、較多的細胞板狀偽足的延展和最快的成長速率，在100和200的奈米點上發現細胞有類似凋亡和貼附面積明顯減少的現象產生。利用免疫螢光染色探討細胞vinculin和actin filament在奈米表面的分佈，發現50奈米點會促進細胞貼附和細胞骨架的形成，在100和200奈米點會阻礙焦點附著以及細胞骨架的形成。 我們更進一步使用Real-Time RT-PCR對細胞的基因作分析，發現在100以及200奈米點上，細胞分泌Chemokines (CCL-2/MCP-1 and CCL-3/MIP-1α)， Cytokines (IL-10, TNF-α, and IL-6) 與貼附分子(VEGF)有明顯的增加，在50奈米點上，細胞表現較高的PAI-1，我們推測100和200的奈米點會誘發細胞產生發炎反應、氧化壓力以及凋亡。最後利用Western blot來分析蛋白質的表現，細胞在50奈米點上有明顯的vinculin和PAI-1的表現，結果顯示50奈米點會促進細胞貼附良好且會抑制細胞纖維蛋白溶解。 實驗結果顯示利用奈米點陣列來調控小鼠肺腫瘤細胞生長與尺寸大小有關。細胞在50奈米點上具有最佳的存活率、型態、貼附和極細微的纖維蛋白溶解；細胞生長在100奈米以上的奈米點表面會阻礙細胞的生長情形與細胞板狀偽足的延展。往後可以利用此研究的結果得知表面結構的重要性在於對細胞行為有顯著的影響，我們的裝置將會在組織工程與癌症治療上扮演一個快速又方便的工具。

Biomaterials play an important role in bioengineering applications. To evaluate cellular response to a nanoscaled surface, Mouse Lewis Lung Carcinoma (LL/2) cells 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. The size-dependent effect of Nanodot arrays on cell growth, corresponding to cell proliferation, cell adhesion, cytoskeletal organization, and gene expression is the main point of our study. Optimized growth occurred when LL/2 cells were cultured on nanodot arrays with dot size at 50-nm, on which maximum viability was maintained even when the cell density reached saturation. Nanodots of 100 and 200-nm prevented viable growth of LL/2 cells with 34.7% and 46.71% reduction at 72 hours, respectively. Cells seeded on 50-nm nanodots showed cell morphology with largest surface area, more extended lamellipodia, and fastest growth rate. Apoptosis-like growth was observed on 100 and 200-nm nanodots with significant reduction in the surface area. Immunostaining was performed against vinculin and actin filaments indicated that 50-nm nanodots promote cell adhesion and cytoskeletal organization. 100-nm nanodots retarded the formation of focal adhesion while 200-nm nanodots inhibited the organization of cytoskeleton. Then, we carry out an in vitro gene function study using quantitative Real-Time RT-PCR. Levels of mRNAs encoding Chemokines (CCL-2/MCP-1 and CCL-3/MIP-1α), Cytokines, (IL-10, TNF-α, and IL-6) and Adhesion molecules (VEGF) increased significantly on the 100 and 200-nm nanodots. The mRNA encoding fibrinolysis molecule (PAI-1) has high expression on 50-nm nanodots. The most cells seeded on 100 and 200-nm nanodots induced inflammatory responses, oxidative stress, and aopotosis. Finally, utilizing Western blot analyzed protein expression. High expression of vinculin and PAI-1 were occurred on 50-nm diameter nanodots. It indicated that 50-nm nanodots have good focal adhesion and suppress fibrinolysis with cells. The results presented here illustrate the ability of nanodot arrays to modulate the growth of LL/2 cells is size-dependent. Optimized growth with the best viability, morphology, adhesion, and imperceptible fibrinolysis occurred at size of 50-nm. Retardation of growth and lamellipodia extension were observed when the dot size was larger than 100-nm. The important of a surface characterization may have a significant effect on cellular behavior. Our device will serve as a convenient and fast tool for tissue engineering and cancer treatment.