Engineering of Artificial Biomatrix through Extracellular Matrix Targeting of Functional Proteins

通过功能蛋白的细胞外基质靶向进行人工生物基质工程

• 批准号: 11558081
• 负责人: SEKIGUCHI Kiyotoshi
• 金额: $ 6.34万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11558081/
• 关键词: Extracellular matrix; basement membrane; matrix engineering; growth factor; wound healing; laminin; cell adhesion; HGF; インテグリン; フィブロネクチン

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

利用蛋白靶向细胞外基质的方法制备人工生物基质。首先，我们建立了一种方案，将转化生长因子- α (TGF-a)通过与纤维连接蛋白的基质组装域的嵌合靶向到纤维连接蛋白基质上。所得到的嵌合蛋白保留了TGF-a的有丝分裂活性，而不影响其基质组装活性。该嵌合蛋白在促进兔角膜愈合方面的作用优于原可溶性TGF-a，显示了其作为一种促进创面愈合的新型蛋白药物的潜力。其次，我们开发了另一种方案，将功能蛋白靶向基膜，这是干细胞生长和分化的支架。我们利用agrin的n端层粘连蛋白结合域将肝细胞生长因子（HGF）或绿色荧光蛋白靶向到基膜上。所得到的嵌合蛋白不仅保留了HGF的生运动活性，而且还保留了agrin的层粘连蛋白结合活性。当表达嵌合蛋白的细胞在含有层粘连蛋白的基底膜样凝胶（即Matrigel）上生长时，发现嵌合蛋白沉积在细胞周围。本方案将适用于各种生长因子和/或细胞分化诱导因子靶向血管基底膜和其他器官。第三，我们纯化了新的层粘连蛋白异构体，即层粘连蛋白-8和-10，并对它们的生理活性进行了表征，特别参考了整合素介导的信号事件。用cDNA转染人293细胞，建立了层粘连蛋白-6、-8和-10的表达体系。我们还成功地表达了层粘连蛋白链的功能区域，并推测其具有整合素/ α -三磷酸腺苷结合以及层粘连蛋白自组装的活性。层粘连蛋白链的这些结构域将有助于构建适合特定细胞类型（如胚胎和组织干细胞）增殖和分化的量身定制的人工基底膜。少

项目成果

Satoi, S., Hiramatsu, Y., Kitade, H., Kwon, A.-H., Matsui, K., Miyashita, K., Sakashita, E., Sekiguchi, K., Takahashi, H., and Kamiyama, Y.: "Different responses to surgical stress between extra domain A+ and plasma fibronectin"Clin. Exp. Pharmcol. Physio

佐井 S.、平松 Y.、北出 H.、权 A.-H.、松井 K.、宫下 K.、坂下 E.、关口 K.、高桥 H. 和神山

Fujiwara, H., et al.: "Purification and characterization of human laminin-8;laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins"J.Biol.Chem.. 276. 19550-19558 (2001)

Fujiwara, H., 等人：“人层粘连蛋白 8 的纯化和表征；层粘连蛋白 8 通过 α3β1 和 α6β1 整合素刺激细胞粘附和迁移”J.Biol.Chem.. 276. 19550-19558 (2001)

関口 清俊: "多細胞体の構築と細胞接着システム"共立出版. 208 (2001)

Kiyotoshi Sekiguchi：“多细胞体和细胞粘附系统的构建”Kyoritsu Shuppan 208（2001）。

Fujiwara,H., Kikkawa,Y., Sanzen,N., and Sekiguchi,K.: "Purification and characterization of human laminin-8: laminin-8 stimulates cell adhesion and migration through α3β1 and α6β1 integrins"J. Biol. Chem.. 276. 17550-17558 (2001)

Fujiwara, H.、Kikkawa, Y.、Sanzen, N. 和 Sekiguchi, K.：“人层粘连蛋白 8 的纯化和表征：层粘连蛋白 8 通过 α3β1 和 α6β1 整合素刺激细胞粘附和迁移” J. Biol。 ..276.17550-17558 (2001)

Fleischmajor, R., Kuroda, K., Utani, A., MacDonald, E. D., Perlish, J. S., Arikawa-Hirasawa, E., Sekiguchi, K., Sanzen, N., Timpl, R., and Yamada, Y.: "Differential expression of laminin α chains during proliferative and differentiation stages of skin mor

Fleischmajor, R.、Kuroda, K.、Utani, A.、MacDonald, E. D.、Perlish, J. S.、Arikawa-Hirasawa, E.、Sekiguchi, K.、Sanzen, N.、Timpl, R. 和 Yamada, Y. ：“皮肤组织增殖和分化阶段层粘连蛋白α链的差异表达