2012-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/655384摘要：細胞於其原生環境中會與其他細胞和細胞外基質相互作用。細胞外基質(ECM) 提供了機械、生化、與地形上的微環境。地形環境會影響細胞的排列及遷移，這種現象被稱為接觸指引(contact guidance)。接觸指引在組織形態發生與癌症轉移扮演很重要的角色。最近比較細胞處於二維和三維空間的研究報告指出兩者之間的一些顯著差異，然而這些研究往往比較硬質玻璃或塑料基板上的2D細胞，與包埋在軟的3D材料中的細胞。我們最近開發了一種經濟且易於使用的微流體仿生平台，以生成具有高度排列的膠原蛋白凝膠，可精確控制細胞於凝膠中的位置以控制機械微環境的參數。在此計畫中，我們推測在3D中的接觸指引造成之細胞形態、細胞骨架和遷移的改變是由RhoA調停控制；我們將使用我們的仿生平台，以調查細胞在二維和三維系統的形態和遷移的基本機制。首先將研究纖維細胞在二維和三維系統中的接觸指引行為，並了解每一個細胞骨架成分的角色。然後將研究地形對RhoA的活性和細胞的張力平衡的影響。此外將調查相關的RhoA信號通路。本計畫將首次直接比較細胞在二維和三維的行為，其結果將增近微環境對細胞調節的機制的了解，並有助於發育生物學、再生醫學、與腫瘤轉移的研究。<br> Abstract: Cells in their native environment reside in a social context where they interact with other cells and the extracellular matrix (ECM). The ECM provides a mechanical, biochemical, and topographical environment. The topographical cues direct cell alignment and migration, and this phenomenon is termed contact guidance. Contact guidance is important in tissue morphogenesis and cancer metastasis. Studies done on planar substrates (2D) showed that cell migration is controlled by contraction-mediated focal adhesion, Rho activation, and cytoskeleton organization. Furthermore, recent reports comparing 2D and 3D studies have identified a number of significant differences in cell adhesion structure and cytoskeleton organization. However, these studies often compare cells seeded on rigid glass or plastic substrates with cells embedded in a soft 3D material. We recently developed an economical and easy-to-use microfluidic platform to generate thin collagen hydrogels with highly aligned collagen fibers (aligned collagen hydrogel, ACH). The hydrogel is attached to a glass slide, with precisely controlled gel thickness and cell position that allows independent variation of these parameters. In this application, we hypothesize that RhoA is a central mediator of 3D contact guided behaviors, including cell morphology, cytoskeleton organization, and migration. We propose to use our biomimetic platform to investigate the underlying mechanisms behind the contact guided morphology and migration in 2D and 3D systems. We propose to first characterize the contact guided behaviors of fibroblasts in our aligned collagen hydrogel and the roles of each cytoskeletal components. We will then examine the effects of substrate topography on RhoA activities and cell tensional balance. Furthermore, we will investigate the related signaling pathways. Taking advantage of microfluidic technology, this proposal will for the first time directly compare cell behaviors in 2D and 3D without changes in neither chemical nor physical properties. Findings from this project will improve the current understanding of microenvironmental regulation of cell morphology and migration, which has great implications in developmental biology, regenerative medicine, and cancer metastasis research.