奈米表面與時序調控人類類造骨細胞整合蛋白的組合

Abstract

主旨為了解在不同的奈米尺度與時間造成的組合蛋白表現差異以及其單元組合的變化。透過本研究明確地了解空間以及時間的變化如何影響組合蛋白的表現，並透過空間的挪移和時間的演進，觀察組合蛋白的組合變化，遂將MG63人類類造骨細胞培養在flat, 10 nm, 50 nm, 100 nm, and 200 nm的奈米點陣列上。 實驗結果顯示，在細胞型態的實驗中，類造骨細胞培養在flat, 10 nm, 和 50 nm的表面，其細胞型態完整、細胞體較大且貼附狀況良好，其中又以50 nm的表面，細胞體積的體現相對其他表面來的突出。加以，在螢光染色實驗中，Vinculin和actin filament的分布探討奈米表面對細胞focal adhesion以及骨架的變化，進而從細胞focal adhesion localization的狀況觀察到Flat, 10 nm, 和 50 nm的表面會促進細胞的貼附和細胞骨架的完整。 為了探討組合蛋白的表現差異，以及其組合蛋白之組合變化。在基因表現方面，組合蛋白的表現高峰出現在時間點12和24小時，而偏好表現在flat, 10 nm, 和50 nm的表面上。在組合蛋白的單元配對(α單元與β單元)，flat與10 nm相似性高，主要的整合蛋白組合各為αvβ3, 和αvβ5；50 nm則自成一格，其組合為αvβ3和αvβ5；而100 nm和200 nm除了表現量都偏低之外，單元配對的組成的種類也比flat, 10 nm, 和50 nm組別單純，100 nm和200 nm的組合各為αvβ3和α8β1。在蛋白表現的結果除了再次證明組合蛋白的表現受到時間與奈米尺度的影響，也發現組合蛋白的表現有時間延遲的狀況。 整個結果顯示，Flat, 10 nm, 和 50 nm結構表面有利於我們觀察組合蛋白在空間以及時間上的變化外，還有助於我們探討組合蛋白之組合替換的相關機制，再者，其表現出的組合蛋白多樣性甚至可作為未來在掌控細胞行為一個很好的工具。最後，也期望本研究能夠在人工骨植入之生物醫學的領域提供貢獻。

Aiming is to investigate that how the different nanodos array and time frames influence the integrins expression and associated combination about the integrin heterodimers. Through our studies to exactly understand that how the nanoscale surfaces regulate the integrin subunits expression and the heterodimers grouping. Then, the human osteoblast-like cells were cultured on the flat, 10 nm, 50 nm, 100 nm, and 200 nm nanodots array. Cell cultured on flat, 10 nm, and 50 nm, which cell morphology were integrated, larger cell area, and better cell attachment than 100 nm, and 200 nm can be observed. Through the distribution of vinculin and actin filament is in order to investigate the nanotopogrphic modulated-cell focal adhesion and reorganization of cytoskeleton. By the results, the cell focal adhesion protein localization demonstrated the promoting cell attachment and integrated cell cytoskeleton are contributed to the nanosurfaces, flat, 10 nm, and 50 nm. In order to study integrins expression and their pairs, the integrin genes expression results exhibited that the significant genes expression of integrin has shown in 12 and 24 hours with the preferred surfaces, flat, 10 nm, and 50 nm. In the pairing of the integrin heterodimers (α and β subunits), although the high similarities has displayed on the flat and 10 nm, 50 nm possessed the different combinations. Finally, in addition to the lower expression of integrins, the pairs on the100 nm, and 200 nm were relatively simplification. In the proteins level, which demonstrated the topologic and temporal controlled- integrin pairing, and also discovered the time-delayed about the integrin proteins level, which might be associated with the mechanism of degradation and recycling of integrins. In general, flat, 10 nm, and 50 nm scales were available in researching the topologic- and temporal-modulating pairing of integrins, and suitable in investigate the mechanism of integrin heterodimers replacing. Furthermore, the integrin varieties provide us another suggestion in controlling the cell behavior. Finally, expecting this study will contribute to the researches in the implant therapies of biomedicine.