Kobayashi Jun, Arisaka Yoshinori, Yui Nobuhiko, Yamato Masayuki, Okano Teruo
Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns 8-1 Kawadacho, Shinjuku-ku Tokyo 162-8666 Japan
Department of Organic Biomaterials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University 2-3-10 Kanda-Surugadai, Chiyoda-ku Tokyo 101-0062 Japan.
RSC Adv. 2021 Nov 18;11(59):37225-37232. doi: 10.1039/d1ra07317f. eCollection 2021 Nov 17.
A heparin-modified poly(-isopropylacrylamide) (PIPAAm)-grafted surface bound with heparin-binding epidermal growth factor-like growth factor (HB-EGF) was able to culture hepatocytes maintaining high albumin secretion and high expression of hepatocyte-specific genes. However, the activity of HB-EGF on the surface and its binding effects on hepatocytes remain unclear. In this study, we investigated the temperature-dependent interactions of HB-EGF and EGF receptor (EGFR) with heparin-modified PIPAAm to evaluate the activity of HB-EGF on the surface. Quartz crystal microbalance (QCM) measurements revealed that the amounts of adsorbed HB-EGF on either the heparin-modified PIPAAm-grafted surface (heparin-IC1) or PIPAAm-grafted surfaces were almost the same regardless of swelling/deswelling of grafted PIPAAm chains. The heparin-IC1 surface bound to HB-EGF at 37 °C had the ability to bind to hepatocytes through specific affinity interaction with EGFR, whose activation was confirmed by western blotting. However, the physisorbed HB-EGF on the PIPAAm surface greatly diminished its activity. Taken together, the introduction of heparin into grafted PIPAAm chains on the surface plays a pivotal role in holding HB-EGF while preserving its activity. Hydration and swelling of surface-grafted PIPAAm chains at 20 °C greatly diminished the attachment of hepatocytes with HB-EGF bound to heparin-IC1, whereas hepatocytes were able to bind to HB-EGF bound to heparin-IC1 at 37 °C. Thus, the equilibrated affinity interaction between EGFRs and surface-bound HB-EGF was considered to be attenuated by steric hindrance due to hydration and/or swelling of grafted PIPAAm chains.
结合有肝素结合表皮生长因子样生长因子（HB - EGF）的肝素修饰聚（N - 异丙基丙烯酰胺）（PIPAAm）接枝表面能够培养肝细胞，维持高白蛋白分泌和肝细胞特异性基因的高表达。然而，表面上HB - EGF的活性及其对肝细胞的结合作用仍不清楚。在本研究中，我们研究了HB - EGF和表皮生长因子受体（EGFR）与肝素修饰的PIPAAm之间的温度依赖性相互作用，以评估表面上HB - EGF的活性。石英晶体微天平（QCM）测量显示，无论接枝的PIPAAm链的溶胀/去溶胀情况如何，肝素修饰的PIPAAm接枝表面（肝素 - IC1）或PIPAAm接枝表面上吸附的HB - EGF量几乎相同。在37℃与HB - EGF结合的肝素 - IC1表面能够通过与EGFR的特异性亲和相互作用与肝细胞结合，其激活通过蛋白质印迹法得到证实。然而，PIPAAm表面上物理吸附的HB - EGF大大降低了其活性。综上所述，在接枝的PIPAAm链中引入肝素在保持HB - EGF活性的同时对其固定起着关键作用。表面接枝的PIPAAm链在20℃时的水合和溶胀极大地减少了肝细胞与结合到肝素 - IC1上的HB - EGF的附着，而肝细胞在37℃时能够与结合到肝素 - IC1上的HB - EGF结合。因此，EGFR与表面结合的HB - EGF之间的平衡亲和相互作用被认为由于接枝的PIPAAm链的水合和/或溶胀导致的空间位阻而减弱。
