Ito Y, Chen G, Imanishi Y
Department of Material Chemistry, Faculty of Engineering, Kyoto University, Japan.
Bioconjug Chem. 1998 Mar-Apr;9(2):277-82. doi: 10.1021/bc970190b.
Photoreactive epidermal growth factor (EGF) was synthesized by conjugating mouse EGF with photoreactive polyallylamine, which was synthesized by the coupling reaction of polyallylamine with N-[4-(azidobenzoyl)oxy]succinimide. The EGF derivative was pattern-immobilized onto a polystyrene plate by UV irradiation in the presence of a photomask in a prescribed micropattern. The patterned immobilization of EGF on the polystyrene plate was confirmed by immunostaining with anti-EGF antibody. Chinese hamster ovary (CHO) cells overexpressing EGF receptors were cultured on the micropatterned plate. The phosphorylated tyrosine residues of signal proteins, including EGF receptors, were detected only in the cells adhered in the EGF-immobilized area, and cell growth was observed only in the EGF-immobilized area. The cells growing in the EGF-immobilized area were partially stained by anti-phosphotyrosine antibody, when the area of EGF immobilization was smaller than the cell. The partial staining of activated proteins indicates that immobilization of EGF inhibited the free lateral diffusion and internalization of the activated EGF-EGF receptor complex. The enhanced cell growth is due to juxtacrine stimulation realized by immobilized EGF.
通过将小鼠表皮生长因子（EGF）与光反应性聚烯丙胺偶联来合成光反应性表皮生长因子，其中光反应性聚烯丙胺是通过聚烯丙胺与N-[4-(叠氮苯甲酰基)氧基]琥珀酰亚胺的偶联反应合成的。在光掩模存在下，通过紫外线照射在规定的微图案中将EGF衍生物图案化固定在聚苯乙烯板上。通过用抗EGF抗体进行免疫染色来确认EGF在聚苯乙烯板上的图案化固定。将过表达EGF受体的中国仓鼠卵巢（CHO）细胞培养在微图案化板上。仅在粘附于EGF固定区域的细胞中检测到包括EGF受体在内的信号蛋白的磷酸化酪氨酸残基，并且仅在EGF固定区域观察到细胞生长。当EGF固定区域小于细胞时，在EGF固定区域生长的细胞被抗磷酸酪氨酸抗体部分染色。活化蛋白的部分染色表明EGF的固定抑制了活化的EGF-EGF受体复合物的自由侧向扩散和内化。细胞生长增强是由于固定化EGF实现的旁分泌刺激。
