Carvalho Vera, Domingues Lucília, Gama Miguel
IBB, Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.
Mol Biotechnol. 2008 Nov;40(3):269-79. doi: 10.1007/s12033-008-9089-9. Epub 2008 Aug 2.
Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation, and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). A method based on the use of a carbohydrate-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial.A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyze the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitin-binding domain.
用于组织工程应用的生物材料必须为组织发育提供结构支撑，并且还能与细胞积极相互作用，促进细胞黏附、增殖和分化。为实现这一目标，可以使用黏附分子，比如三肽精氨酸 - 甘氨酸 - 天冬氨酸（RGD）。一种基于使用对几丁质具有亲和力的碳水化合物结合模块的方法，作为生物活性肽化学接枝的替代方法进行了测试。这种方法将同时允许生产重组肽（替代肽合成），并为肽特异性且牢固地吸附到生物材料上提供一种简单方式。一种融合重组蛋白在大肠杆菌中表达，该蛋白包含与人几丁质结合模块（ChBM）融合的RGD序列。成纤维细胞与再乙酰化壳聚糖（RC）膜的黏附是被选用来分析所得蛋白质特性的模型系统。因此，使用小鼠胚胎成纤维细胞3T3对涂覆有重组蛋白的聚苯乙烯表面和再乙酰化壳聚糖膜上的细胞附着和增殖进行了评估。结果表明，重组蛋白对成纤维细胞在材料表面的锚定产生负面影响，抑制其黏附和增殖。我们还得出结论，这种负面影响主要归因于人类几丁质结合结构域。
