Engineering of Artificial Biomatrix through Extracellular Matrix Targeting of Functional Proteins

已经开发了通过将蛋白质靶向至细胞外基质来工程化人工生物基质的新方案。首先，我们建立了一个方案，通过嵌合与纤连蛋白的基质组装结构域的靶向转化生长因子-α（TGF-α）的纤连蛋白基质。所得嵌合蛋白保留TGF-α的促有丝分裂活性而不损害其基质组装活性。嵌合蛋白在促进兔角膜伤口愈合方面比真实的可溶性TGF-α更有效，证明其作为加速伤口愈合的新蛋白质药物的效力。其次，我们开发了另一种方案，将功能蛋白质靶向基底膜，即干细胞生长和分化的支架。我们利用聚集蛋白的N-末端层粘连蛋白结合结构域将肝细胞生长因子（HGF）或绿色荧光蛋白靶向基底膜。所得到的嵌合蛋白保留了HGF的运动原活性 ...更多信息 以及聚集蛋白的层粘连蛋白结合活性。当表达嵌合蛋白的细胞在含层粘连蛋白的基底膜样凝胶上生长时（即，Matrigel），发现嵌合蛋白沉积在细胞周围。本方案将适用于将各种生长因子和/或细胞分化诱导因子靶向血管和其他器官的基底膜。第三，我们纯化了新的层粘连蛋白同种型，即，层粘连蛋白-8和-10，并通过特别参考整合素介导的信号传导事件来表征它们的生理活性。我们还通过cDNA转染人293细胞建立了层粘连蛋白-6、-8和-10的表达系统。我们还成功地表达了层粘连蛋白链的功能结构域，其具有整合素/α-肌营养不良蛋白聚糖结合以及层粘连蛋白自组装的推定活性。层粘连蛋白链的这些结构域将可用于构建针对特定细胞类型的增殖和分化而优化的定制的人工基底膜，胚胎和组织干细胞。少

Novel protocols for engineering of artificial biomatrix by targeting of proteins to the extracellular matrices have been developed. First, we established a protocol to target transforming growth factor-alpha (TGF-a) to the fibronectin matrix via chimerization with the marix assembly domain of fibronectin. The resulting chimeric protein retains the mitogenic activity of TGF-a without-compromising its matrix assembly activity. The chimeric protein was more potent than authentic, soluble TGF-a in promoting would healing of rabbit cornea, demonstrating its potency as a new protein drug for accelerating, wound healing. Second, we developed another protocol to target functional proteins to the basement membrane, the scaffold for growth and differentiation of stem cells. We utilized the N-terminal laminin-binding domain of agrin to target hepatocyte growth factor (HGF) or green fluorescent protein to the basement membrane. The resulting chimeric protein retained the motogenic activity of HGF … More as well as the laminin-binding activity of agrin. When cells expressing the chimeric protein were grown on the laminin-containing basement membrane-like gel (I.e., Matrigel), the chimeric protein was found to be deposited around the cells. This protocol will be applicable for targeting of various growth factors and/or cell differentiation-inducing factors to the basement membranes of vasculature and other organs. Third, we purified new laminin isoforms, I.e., laminin-8 and -10, and characterized their physiologic activities with special reference to integrin-mediated signaling events. We also established the expression system for laminin-6, -8, and -10 by cDNA transfection into human 293 cells. We also succeeded in expression of the functional domains of laminin chains with putative activities for integrin/alpha-dystroglycan binding as well as laminin selfassembly. These domains of laminin chains will be useful for construction of tailor-made, artificial basement membranes optimized for proliferation and differentiation of particular cell types, e.g., embryonic and tissue stem cells. Less