奈米孔洞拓樸結構透過調節integrins和ERK的表現

Abstract

Integrin調控focal adhesion的形成對於細胞貼附至外在環境上是不可或缺的，奈米結構可控制許多種細胞行為，例如:細胞的貼附與移動。然而，表面結構影響細胞行為的反應機制目前還未研究透徹。本研究將探討纖維母細胞在40至210奈米的316L不鏽鋼奈米孔洞上的行為表現，包括:細胞的生長、移動能力與integrins和ERK的調控。 經實驗結果發現纖維母細胞在直徑40和75奈米孔洞結構上培養24小時後，可促進細胞增生、貼附及良好的細胞型態。我們更進一步使用qPCR分析integrin於奈米孔洞上的表現，其結果顯示integrin會受到空間及時間性的調控。西方墨點法的分析顯示在40和75奈米上培養24小時後，vinculin，β-tubulin，integrin αV和α2會有明顯的表現。而ERK1/2的磷酸化在185和210奈米上培養12和24小時後，其表現量明顯較其它尺寸低。最後利用wound healing分析細胞的移動，當細胞在40和75奈米孔洞上培養12小時後，顯示有較好的移動能力。 實驗結果顯示40和75奈米孔洞結構可促進細胞的貼附、移動並調控integrin、ERK和貼附相關分子的表現。我們目前的研究發現奈米結構可藉由空間及時間的影響，來控制細胞行為與分子的表現，而這些實驗參數則可用以改善不鏽鋼植入物的生物相容性。

The integrin-mediated formation of focal adhesions is essential for the adhesion of migrating cells to a substrate. The nanotopography controls the cell’s behaviors, such as cell adhesion and migration. However, the mechanisms responsible for topology-mediated cellular functions are not fully understood. A variety of nanopores fabricated on 316L stainless steel were applied to investigate the effects of spatial control on the growth and function of fibroblasts, the temporal regulation of integrins and ERK, and their effects on the migration. The fibroblast cell line NIH 3T3 was cultured on nanopore surfaces with pore diameters ranging from 40 to 210 nm. The 40 and 75 nm nanopores enhanced cell proliferation and focal adhesion formation after 24 h incubation, with a large cell spreading area was observed. Integrin expression was analyzed by qPCR, which showed their spatial and temporal regulation by the nanopores. Western blot analysis verified high levels of expression of vinculin, β-tubulin, integrin αV, and integrin α2 for cells cultured on 40 and 75 nm nanopores starting from 24 h. The protein expression of pERK1/2 was greatly attenuated in cells grown on 185 and 210 nm at 12 and 24 h. Significantly enhanced cell migration was also observed for fibroblasts grown on 40 and 75 nm nanopores after 12 h of incubation in a wound healing assay. In summary, the 40 and 75 nm nanopore surfaces promoted cell adhesion and migration in fibroblasts by controlling the temporal expression of integrins and focal adhesion molecules. We found that the nanostructure could control cell behaviors and molecular expressions by spatial and temporal regulation. The current study provides insight to improve the design of stainless steel implants and parameters that affect biocompatibility.