Iwasaki Yasuhiko, Matsunaga Aki, Fujii Shuetsu
Department of Chemistry and Materials Engineering Faculty of Chemistry, Materials and Bioengineering, Kansai University , 3-3-35 Yamate-cho, Suita-shi, Osaka 564-8680, Japan.
Bioconjug Chem. 2014 Sep 17;25(9):1626-31. doi: 10.1021/bc5003295. Epub 2014 Aug 21.
In the current study, synthetic hydrogels containing metabolically engineered glycoproteins of mammalian cells were prepared for the first time and selectin-mediated cell adhesion on the hydrogel was demonstrated. A culture of HL-60 cells was supplemented with an appropriate volume of aqueous solution of N-methacryloyl mannosamine (ManMA) to give a final concentration of 5 mM. The cells were then incubated for 3 days to deliver methacryloyl groups to the glycoproteins of the cells. A transparent hydrogel was formed via redox radical polymerization of methacryloyl functionalized glycoproteins with 2-methacryloyloxyethyl phosphorylcholine and a cross-linker. Conjugation of the glycoproteins into the hydrogel was determined using Coomassie brilliant blue (CBB) and periodic acid-Schiff (PAS) staining. The surface density of P-selectin glycoprotein ligand-1 (PSGL-1) on the hydrogels was also detected using gold-colloid-labeled immunoassay. Finally, selectin-mediated cell adhesion on hydrogels containing glycoproteins was demonstrated. Selectin-mediated cell adhesion is considered an essential step in the progression of various diseases; therefore, hydrogels having glycoproteins could be useful in therapeutic and diagnostic applications.
在本研究中，首次制备了含有经代谢工程改造的哺乳动物细胞糖蛋白的合成水凝胶，并证明了水凝胶上选择素介导的细胞粘附。向HL-60细胞培养物中补充适量体积的N-甲基丙烯酰甘露糖胺（ManMA）水溶液，使其终浓度为5 mM。然后将细胞孵育3天，以将甲基丙烯酰基传递到细胞的糖蛋白上。通过甲基丙烯酰基功能化糖蛋白与2-甲基丙烯酰氧基乙基磷酰胆碱和交联剂的氧化还原自由基聚合形成透明水凝胶。使用考马斯亮蓝（CBB）和过碘酸希夫（PAS）染色来确定糖蛋白与水凝胶的结合。还使用金胶体标记免疫测定法检测水凝胶上P-选择素糖蛋白配体-1（PSGL-1）的表面密度。最后，证明了选择素介导的细胞在含有糖蛋白的水凝胶上的粘附。选择素介导的细胞粘附被认为是各种疾病进展中的一个重要步骤；因此，含有糖蛋白的水凝胶可用于治疗和诊断应用。
