SYNTHESIS AND EVALUATION OF OLIGOPEPTIDES EXHIBITING CELL-ATTACHMENT ACTIVITY FOR TISSUE ENGINEERING USE

用于组织工程用途的具有细胞附着活性的寡肽的合成和评估

• 批准号: 12680854
• 负责人: HIRANO Yoshiaki
• 金额: $ 2.24万
• 依托单位: OSAKA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12680854/
• 关键词: Cell-attachment oligopeptides; RODS; RODS mimetic peptide; Peptide-immobilized materials; Conformational analysis; ECEPP; Inhibition of platelet aggregation; Structure-activity relationship; 細胞接着性オリゴペプチド; RGDSミメチィックペプチド; 生医学材料

Arg-Gly-Asp-Xaa (RGDX) sequence is a cell-adhesion motif present in several matrixassociated adhesive glycoproteins including fibronectin, vitronectin, and fibrinogen. The RGDX sequence is recognized by several integrins, including the platelet, fibroblast cell, and so on. It has been demonstrated that soluble peptides containing the RGD sequence compete with RGDcontaining insoluble matrix proteins for binding to their respective integrins, and thus prevent cellmatrix adhesion. However, the affinity of the short synthetic peptides to their corresponding integrins is lower than that of proteins. In this work, we designed and synthesized Arg-Gly-AspSer (RGDS) mimetic peptides for the purpose of improving the cell attachment activity of RGDS oligopeptide, and their cell-attachment activities were assayed by L929 fibroblast cell toward peptide-immobilized polymeric materials and platelet aggregation inhibition method. Then we discussed on the structure and activity relationship of RGDS mim … More etic peptides.RGDS and its mimetic peptides, and RGDS containing β -sheet model peptides were synthesized using liquid ancl solid phase procedures. All peptides were characterized by NMR, MALDI-TOF MS, amino acid analysis, and elemental analysis. Cell-attachment activities of these peptides were examined by cell- attachment test using L929 fibroblast cell toward peptide-immobilized PVA film.Number of L929 cells attaching to RGDS mimetic peptide-immobilized PVA films at incubation times of 1, 3, 6 and 24 hr. The cell-attachment activity of Har-Gly-Asp-Ser (hRGDS) and Orn-Gly-Asp-Ser (OrGDS) was the same as that of RGDS. However, the cell attachment activity of Arg-Nip-Asp-Ser (RNiDS) was higher than that of RGDS from the initial stage, and cell attachment activity of RNiDS was about eight times as high as that of RGDS after incubation for 24hr. The hRGDS and OrGDS peptides present the same CD spectra as that ofRGDS. The spectral patterns suggested that these peptides possess a type I-β bend. However, RNiDS takes a typical type II-β bend. RNiDS peptide takes the different type of turn structure in comparison with the other RGDS mimetic peptides. It seems that RNiDS forms the optimum conformation for binding to the integrin receptor. It is necessary that for the active expression, the RGDS mimetic peptides take the optimum conformation in the PVA film surface. The nipecotic acid residue of RNiDS appears to contain an important structural motif that contributed to the observed cell-attachment activity. Less

Arg-Gly-Asp-Xaa （RGDX）序列是一个细胞粘附基序，存在于几种基质相关的粘附糖蛋白中，包括纤维连接蛋白、玻璃体连接蛋白和纤维蛋白原。RGDX序列被多种整合素识别，包括血小板、成纤维细胞等。已经证明，含有RGD序列的可溶性肽与含有RGD序列的不溶性基质蛋白竞争，以结合各自的整合素，从而防止细胞基质粘附。然而，合成的短肽对其相应的整合素的亲和力低于蛋白质。本文设计并合成了Arg-Gly-AspSer （RGDS）模拟肽，以提高RGDS寡肽的细胞粘附活性，并采用L929成纤维细胞对肽固定化聚合物材料和血小板聚集抑制法测定了其细胞粘附活性。然后讨论了RGDS与多肽的结构和活性关系。采用液相法和固相法合成了RGDS及其模拟肽，以及含有β -sheet模型肽的RGDS。所有肽均通过NMR， MALDI-TOF MS，氨基酸分析和元素分析进行了表征。利用L929成纤维细胞对肽固定化PVA膜进行细胞附着实验，研究了这些肽对PVA膜的附着活性。孵育1、3、6和24小时时，L929细胞附着在RGDS模拟肽固定化PVA膜上的数量。Har-Gly-Asp-Ser （hRGDS）和Orn-Gly-Asp-Ser （OrGDS）的细胞附着活性与RGDS相同。然而，Arg-Nip-Asp-Ser （RNiDS）的细胞附着活性从一开始就高于RGDS，孵育24小时后，RNiDS的细胞附着活性约为RGDS的8倍。hRGDS和OrGDS肽具有与rgds相同的CD谱。光谱模式表明这些肽具有I-β型弯曲。然而，RNiDS呈现典型的II-β型弯曲。与其它RGDS类肽相比，RNiDS肽具有不同的转型结构。RNiDS似乎形成了与整合素受体结合的最佳构象。RGDS模拟肽在PVA膜表面的最佳构象是活性表达的必要条件。RNiDS的nipecotic酸残基似乎包含一个重要的结构基序，有助于观察到的细胞附着活性。少

项目成果

Masahiro HATTORI, Masahito OKA, Toshio HAYASHI, Yoshiaki HIRANO: "Synthesis and Conformational Analysis of Model Polypeptide Having Repetitve Gly-Pro-Xaa Sequences"Biomedical Materials Research in the Far East. 4. 150-151 (2000)

Masahiro HATTORI、Masahito OKA、Toshio HAYASHI、Yoshiaki HIRANO：“具有重复 Gly-Pro-Xaa 序列的模型多肽的合成和构象分析”远东生物医学材料研究。

T.ISHII, Y.HIRANO, 他3名: "Influence of gelatin complexation on cell proliferation activity and proteolytie resistance of basic fibroblast growth factor"J.Biomaterials Science Polymer Edition. 11. 517-582 (2000)

T.ISHII、Y.HIRANO 和其他 3 人：“明胶络合对碱性成纤维细胞生长因子的细胞增殖活性和蛋白水解抗性的影响”J.Biomaterials Science Polymer Edition 11. 517-582 (2000)。

Y.HIRANO 他5名: "Synthesis of Arg-Gly-Asp-Ser Mimetic Oligopeptides and Evaluation of Their Cell-Attachment Activity"Peptide Science. 2000. 333-336 (2001)

Y.HIRANO 和其他 5 人：“Arg-Gly-Asp-Ser 模拟寡肽的合成及其细胞附着活性的评估”肽科学 2000 年。 333-336 (2001)。

T.IUCH, Y.HIRANO 他4名: "Cell-Attachment Activities of Surface Immobilized RGDS Mimetic Oligopeptides"Peptide Science. 2001(印刷中). (2002)

T.IUCH、Y.HIRANO 和其他 4 人：“表面固定化 RGDS 模拟寡肽的细胞附着活性”，肽科学，2001 年（出版中）。

Yoshiaki Hirano, Masahito Oka, Masahiro Hattori, Toshio Hayashi: "Synthesis and Conformational Analysis of Model Peptides Having Repetitive Xaa-Pro-Pro Sequences"Peptide Science. 1999. 343-346 (2000)

Yoshiaki Hirano、Masahito Oka、Masahiro Hattori、Toshio Hayashi：“具有重复 Xaa-Pro-Pro 序列的模型肽的合成和构象分析”肽科学。