Besselink G A, Beugeling T, Poot A A, Van Aken W G, Bantjes A
Department of Chemical Technology, University of Twente, Enschede, The Netherlands.
J Biomater Sci Polym Ed. 1994;6(8):675-93. doi: 10.1163/156856295x00076.
In this study, affinity adsorbents for the binding of activated blood platelets and endothelial cells were prepared from Sephadex G-10 by immobilization of peptides, derived from the cell-adhesive amino acid sequence RGD (arginine-glycine-aspartic acid). Derivatization of Sephadex G-10 was accomplished by sequential coupling of specific dipeptides (RG and DV) (V = valine) and by coupling of the RGD-containing hexapeptide GRGDSP (S = serine, P = proline). Two types of gel were prepared by sequential coupling, designated as G10 (acetone) and G10 (dimethylformamide) (G10(DMF)), containing peptides which had been coupled to the outer side of the beads and throughout the porous beads, respectively. The binding capacity of the prepared Sephadex-derivatives amounted up to 2 x 10(9) human blood platelets per millilitre GRGDV-Sephadex at immobilized peptide concentrations, that were in the nanomole range (per millilitre packed gel) and which differed a factor 10 between G10 (acetone) and G10 (DMF). In a second series of experiments, different amounts of the hexapeptide GRGDSP were coupled to carboxylated Sephadex G-10 and carboxylated Sepharose CL 6B. The binding of human umbilical vein endothelial cells to the resulting materials was studied. Up to 10(6) endothelial cells attached per ml GRGDSP-derivatized hydrogel at peptide concentrations of 15 nmol GRGDSP/ml Sephadex and at +/- 300 nmol GRGDSP/ml Sepharose. Substitution of the arginine residue of the RGD-sequence by glutamine abolished the cell-binding activity of the immobilized peptide towards activated blood platelets but not towards endothelial cells. From the results of this study it can be concluded that small peptides can be coupled to the outer side of the porous Sephadex beads, resulting in high effective ligand densities for cell-affinity applications. In this respect, Sephadex G-10, derivatized according to 'the acetone method' is a good alternative for polystyrene and other solid phase materials.
在本研究中，通过固定源自细胞黏附氨基酸序列RGD（精氨酸 - 甘氨酸 - 天冬氨酸）的肽，从葡聚糖凝胶G - 10制备了用于结合活化血小板和内皮细胞的亲和吸附剂。葡聚糖凝胶G - 10的衍生化通过特定二肽（RG和DV）（V = 缬氨酸）的顺序偶联以及含RGD的六肽GRGDSP（S = 丝氨酸，P = 脯氨酸）的偶联来完成。通过顺序偶联制备了两种类型的凝胶，分别命名为G10（丙酮）和G10（二甲基甲酰胺）（G10(DMF)），其中含有的肽分别偶联到了珠子的外侧和贯穿多孔珠子内部。在固定化肽浓度处于纳摩尔范围（每毫升填充凝胶）时，制备的葡聚糖衍生物的结合能力达到每毫升GRGDV - 葡聚糖凝胶2×10⁹个人类血小板，并且G10（丙酮）和G10（DMF）之间相差10倍。在第二系列实验中，将不同量的六肽GRGDSP偶联到羧化葡聚糖凝胶G - 10和羧化琼脂糖CL 6B上。研究了人脐静脉内皮细胞与所得材料的结合情况。在肽浓度为15 nmol GRGDSP/毫升葡聚糖凝胶以及±300 nmol GRGDSP/毫升琼脂糖时，每毫升GRGDSP衍生化水凝胶上附着的内皮细胞多达10⁶个。将RGD序列中的精氨酸残基用谷氨酰胺取代，消除了固定化肽对活化血小板的细胞结合活性，但对内皮细胞没有影响。从本研究结果可以得出结论，小肽可以偶联到多孔葡聚糖珠子的外侧，从而在细胞亲和应用中产生高效的配体密度。在这方面，按照“丙酮法”衍生化的葡聚糖凝胶G - 10是聚苯乙烯和其他固相材料的良好替代品。
