Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation
Vitronectin supports migratory responses of corneal epithelial cells to substrate bound IGF-I and HGF, and facilitates serum-free cultivation
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DOI：
10.1016/j.exer.2006.08.012
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发表时间：
2006-12-01
影响因子：
3.4
通讯作者：
Harkin, Damien G.
中科院分区：
文献类型：
--
作者：
Ainscough, S. Louise;Barnard, Zeke;Harkin, Damien G.
玻连蛋白（VN）是一种多功能糖蛋白，最为人所知的是其对细胞附着和扩散的影响，但最近已被证明介导细胞对生长因子的反应。泪液膜内VN的存在和角膜上皮细胞上所需受体（α v整联蛋白）的表达表明在眼表面内具有类似作用的潜力。因此，我们研究了VN改变角膜上皮细胞对与眼表面相关的生长因子的代谢（MTT测定）和迁移（跨膜迁移）反应的能力，所述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）。我们的假设是，用VN包被的培养物表面可能选择性地促进对已知结合VN的生长因子的反应，所述生长因子包括EGF、IGF-I（通过IGF结合蛋白）和HGF。当应用于培养基时，观察到对每种生长因子的代谢反应，但未观察到对用VN和/或生长因子预处理的培养塑料的代谢反应。观察到对IGF-I与EGF联合应用的最佳代谢反应。当基底预涂有VN和IGF-I（与IGFBP-3联合应用）或VN和HGF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，并表明整合素/生长因子受体共活化是角膜上皮细胞迁移所需的。在进一步的研究中，发现VN与IGF-1、IGFBP-3和EGF联合应用（均应用于培养塑料和培养基中）支持在不存在血清的情况下角膜缘上皮细胞培养物的建立和连续繁殖，但辐照的3T3细胞（i3T3）仍然是培养扩增所必需的。角蛋白3和p63的所得培养物的免疫细胞化学显示与在当前最佳实践条件下建立的那些表型相似（i3T3，胎牛血清。EGF与胰岛素总之，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的角膜上皮迁移中的作用，并证明VN-生长因子相互作用可用于在无血清条件下制造生物工程眼表组织。(c)2006爱思唯尔有限公司保留所有权利。
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-1, 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-comeal epithelial cell cultures in the absence of serum, but irradiated 3T3 cells (i3T3) were still necessary for culture expansion. Immunocytochernistry 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 bioeneineered ocular surface tissue under serum-free conditions. (c) 2006 Elsevier Ltd. All rights reserved.