Dynamic Substrates for Cell Adhesion & Motility Studies

描述（由申请人提供）： 用于组织工程等应用的合成材料设计受益于对伤口愈合、组织形态发生和生物医学设备同化等临床事件期间发生的细胞-基质粘附相互作用的更多了解。在此类事件的背景下发生的相互作用是动态的，并且参与受体介导的粘附的受体和配体具有高度的空间和时间灵活性。拟议的工作旨在利用从头设计的蛋白质二聚体 DF2t1 开发具有动态粘附特性的改性聚合物水凝胶表面。二聚体的一个蛋白质分子将用共同的细胞结合结构域 RGD 进行功能化；另一个分子将使用多组分异双功能连接方案缀合到聚丙烯酰胺凝胶的表面。表面与培养的牛主动脉内皮细胞(BAEC)的粘附性将通过含有二聚体成分的RGD与其表面上的补体的二聚化来沉淀。将产生均匀的表面和图案化的表面，以提供观察细胞遇到不同粘附性区域以及表面的粘附特性以化学方式“打开”和“关闭”的机会。将使用定量显微镜方法监测动态粘附条件下的细胞对表面特性的反应，以测量单细胞运动、力牵引产生和细胞群迁移。结果将在临床相关过程（如血管生成和血管生成）期间内皮细胞迁移的背景下进行解释。

DESCRIPTION (provided by applicant): Synthetic materials design for applications such as tissue engineering has benefited from increased understanding of the cell-substrate adhesive interactions that occur during clinical events such as wound healing, tissue morphogenesis, and biomedical device assimilation. Interactions that occur in the context of such events are dynamic, and the receptors and ligands involved in receptor-mediated adhesion possess a high degree of spatial and temporal flexibility. The work proposed seeks to develop a modified polymer hydrogel surface with dynamic adhesion properties utilizing a protein dimer, DF2t1, designed de novo. One protein molecule of the dimer will be functionalized with the common cell-binding domain, RGD; the other molecule will be conjugated to the surface of polyacrylamide gels using a multicomponent heterobifunctional linking scheme. Adhesivity of the surface to cultured bovine aortic endothelial cells (BAEC) will be precipitated by dimerization of the RGD containing component of the dimer to its complement on the surface. Uniform surfaces and patterned surfaces will be produced to provide the opportunity to observe cells as they encounter regions of differing adhesivity, and as the adhesive character of the surface is switched "on" and "off" chemically. The cellular response to surface properties under dynamic adhesion conditions will be monitored using quantitative microscopy methods for measuring single cell motility, force traction generation, and cell population migration. The results will be interpreted in the context of endothelial cell migration during clinically relevant processes such as vasculogenesis and angiogenesis.