Ainscough S Louise, Barnard Zeke, Upton Zee, Harkin Damien G
School of Life Sciences and Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, QLD, Australia.
Exp Eye Res. 2006 Dec;83(6):1505-14. doi: 10.1016/j.exer.2006.08.012. Epub 2006 Oct 16.
Vitronectin (VN) is a multi-functional glycoprotein best known for its effects on cell attachment and spreading, but has more recently been shown to mediate cellular responses to growth factors. The presence of VN within the tear film and expression of required receptors (alpha v integrins) on corneal epithelial cells suggests the potential for a similar role within the ocular surface. Thus we have studied the ability of VN to alter the metabolic (MTT assay) and migratory (trans-membrane migration) responses of corneal epithelial cells to growth factors associated with the ocular surface including epidermal growth factor (EGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF) and insulin-like growth factor-I (IGF-I). Our hypothesis was that culture surfaces coated with VN might selectively facilitate responses to growth factors which are known to bind VN including EGF, IGF-I (via IGF binding protein) and HGF. Metabolic responses were observed towards each growth factor when applied to the culture medium, but not towards culture plastic pre-treated with VN and, or growth factors. Optimal metabolic responses were observed towards IGF-I applied in conjunction with EGF. Migration through porous polycarbonate membrane was significantly increased when the substrate had been pre-coated with VN and IGF-I (applied in conjunction with IGFBP-3) or VN and HGF. This finding is consistent with the ability of IGF-I (via an IGFBP) and HGF to form complexes with VN and suggests that integrin/growth factor receptor co-activation is required for corneal epithelial cell migration. In further studies, VN applied in conjunction with IGF-I, IGFBP-3 and EGF (both to the culture plastic and in the culture medium) was found to support the establishment and serial propagation of limbal-corneal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochemistry of resulting cultures for keratin 3 and p63 revealed a similar phenotype to those established under current best-practice conditions (i3T3, foetal bovine serum, EGF and insulin). In conclusion, our novel findings suggest a role for VN-growth factor complexes in stimulating corneal epithelial migration within the provisional wound bed and demonstrate that VN-growth factors interactions can be exploited to enable manufacture of bioengineered ocular surface tissue under serum-free conditions.
玻连蛋白（VN）是一种多功能糖蛋白，因其对细胞黏附和铺展的作用而最为人所知，但最近已被证明可介导细胞对生长因子的反应。泪膜中存在VN以及角膜上皮细胞上所需受体（αv整合素）的表达表明其在眼表可能具有类似作用。因此，我们研究了VN改变角膜上皮细胞对与眼表相关的生长因子（包括表皮生长因子（EGF）、肝细胞生长因子（HGF）、角质形成细胞生长因子（KGF）和胰岛素样生长因子-I（IGF-I））的代谢（MTT法）和迁移（跨膜迁移）反应的能力。我们的假设是，涂有VN的培养表面可能会选择性地促进对已知能结合VN的生长因子的反应，包括EGF、IGF-I（通过IGF结合蛋白）和HGF。当将每种生长因子应用于培养基时，可观察到对其的代谢反应，但对用VN和/或生长因子预处理的培养塑料则无反应。对与EGF联合应用的IGF-I观察到最佳代谢反应。当底物预先用VN和IGF-I（与IGFBP-3联合应用）或VN和HGF包被时，通过多孔聚碳酸酯膜的迁移显著增加。这一发现与IGF-I（通过IGFBP）和HGF与VN形成复合物的能力一致，并表明角膜上皮细胞迁移需要整合素/生长因子受体共激活。在进一步的研究中，发现将VN与IGF-I、IGFBP-3和EGF联合应用（既应用于培养塑料又应用于培养基中）可在无血清条件下支持角膜缘角膜上皮细胞培养物的建立和连续传代，但培养物扩增仍需要经辐射的3T3细胞（i3T3）。对所得培养物进行角蛋白3和p63的免疫细胞化学分析显示，其表型与在当前最佳实践条件下（i3T3、胎牛血清、EGF和胰岛素）建立的培养物相似。总之，我们的新发现表明VN-生长因子复合物在刺激临时伤口床内角膜上皮迁移中起作用，并证明VN-生长因子相互作用可被利用来在无血清条件下制造生物工程化眼表组织。
