Li J S, Ito Y, Zheng J, Takahashi T, Imanishi Y
Department of Medical Informatics, Faculty of Medicine, Kyoto University, Japan.
J Biomed Mater Res. 1997 Nov;37(2):190-7. doi: 10.1002/(sici)1097-4636(199711)37:2<190::aid-jbm7>3.0.co;2-m.
Insulin was co-immobilized with a cell adhesion factor--fibronectin or polyallylamine--on a surface-hydrolyzed poly(methyl methacrylate) film. Chinese hamster ovary cells overexpressing human insulin receptors were cultured on the film in the absence of serum or soluble proteins. While insulin immobilization did not affect cell adhesion, insulin immobilized on fibronectin-immobilized film reduced the adhesion. Addition of the tetrapeptide Arg-Gly-Asp-Ser (RGDS) inhibited cell adhesion onto fibronectin-immobilized films while cell adhesion onto polyallylamine-immobilized films was not inhibited by RGDS. Small amounts of immobilized insulin (1 to 10% of the amount of free insulin required to achieve cell growth acceleration) were sufficient to stimulate cell proliferation. The maximal mitogenic effect of immobilized insulin was greater than that of free insulin. In addition, co-immobilization with the adhesion factor remarkably enhanced the mitogenic effect. The phosphorylation of the receptor with free insulin attained the maximum degree very rapidly but ceased quickly. On the other hand, the receptor phosphorylation with immobilized insulin was accompanied by a longer induction period and lasted a longer period of time than that with free insulin. Insulin co-immobilization on fibronectin or polyallylamineimmobilized films reduced the induction period by enhancement of cell adhesion. The early and long-lasting receptor activation might have been caused by the greater mitogenic effect of co-immobilized biosignaling polypeptides.
胰岛素与一种细胞黏附因子——纤连蛋白或聚烯丙胺——共同固定在表面水解的聚甲基丙烯酸甲酯薄膜上。在无血清或可溶性蛋白质的条件下，将过表达人胰岛素受体的中国仓鼠卵巢细胞培养在该薄膜上。虽然胰岛素固定化不影响细胞黏附，但固定在纤连蛋白固定化薄膜上的胰岛素会降低黏附。添加四肽Arg-Gly-Asp-Ser（RGDS）可抑制细胞黏附到纤连蛋白固定化薄膜上，而细胞黏附到聚烯丙胺固定化薄膜上不受RGDS抑制。少量固定化胰岛素（达到细胞生长加速所需游离胰岛素量的1%至10%）就足以刺激细胞增殖。固定化胰岛素的最大促有丝分裂作用大于游离胰岛素。此外，与黏附因子共同固定化显著增强了促有丝分裂作用。游离胰岛素使受体磷酸化很快达到最大程度，但很快停止。另一方面，固定化胰岛素使受体磷酸化伴随着较长的诱导期，且持续时间比游离胰岛素长。胰岛素与纤连蛋白或聚烯丙胺固定化薄膜共同固定化通过增强细胞黏附缩短了诱导期。早期且持久的受体激活可能是由共同固定化生物信号多肽的更大促有丝分裂作用引起的。
