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

Arg-Gly-Asp-Xaa(RGDX)序列是一种细胞黏附基序，存在于纤维连接蛋白、玻璃体连接蛋白和纤维蛋白原等多种基质结合的黏附糖蛋白中。RGDX序列可被多种整合素识别，包括血小板、成纤维细胞等。已有研究表明，含有RGD序列的可溶多肽与含有不溶性基质蛋白的RGD竞争结合各自的整合素，从而阻止细胞基质的黏附。然而，短的合成肽与其对应的整合素的亲和力低于蛋白质。为了提高RGDS寡肽的细胞黏附活性，我们设计合成了Arg-Gly-AspSer(RGDS)模拟肽，并用L929成纤维细胞对多肽固定的聚合物材料和血小板聚集抑制法检测了它们的细胞黏附活性。然后讨论了RGDS mim…的构效关系。用液体ANCL固相合成法合成了RGDS及其模拟肽，以及含有β-Sheet模型肽的RGDS。用核磁共振、MALDI-TOF-MS、氨基酸分析和元素分析对其结构进行了表征。用L929成纤维细胞对RGDS模拟多肽固定化PVA膜进行细胞黏附实验，观察1、3、6、24小时后L929细胞对RGDS模拟多肽固定化PVA膜的黏附能力。Har-Gly-Asp-Ser(HRGDS)和Orn-Gly-Asp-Ser(OrGDS)的细胞黏附活性与RGDS相同。而Arg-Nip-Asp-Ser(RNiDS)的细胞黏附活性从培养初期就高于RGDS，培养24小时后RNiDS的细胞黏附活性约为RGDS的8倍。HRGDS和OrGDS具有与RGDS相同的圆二色谱。光谱模式表明这些多肽具有I-β弯曲。然而，RNDS采用典型的II-β弯曲。与其他RGDS模拟肽相比，RNiDS多肽具有不同类型的转角结构。RNiDS似乎形成了与整合素受体结合的最佳构象。对于活性表达，RGDS模拟肽在PVA膜表面具有最佳构象是必要的。RNiDS的胡椒酸残基似乎包含一个重要的结构基序，该基序有助于观察到细胞附着活性。较少

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