Kanayama Shintaro, Nishida Kohji, Yamato Masayuki, Hayashi Ryuhei, Sugiyama Hiroaki, Soma Takeshi, Maeda Naoyuki, Okano Teruo, Tano Yasuo
Department of Ophthalmology, Osaka University Medical School, Suita, Osaka, Japan.
Exp Eye Res. 2007 Dec;85(6):772-81. doi: 10.1016/j.exer.2007.08.011. Epub 2007 Aug 27.
The aim of this study was to compare angiogenesis-induction capabilities of cultured corneal epithelial cells (CCE) and cultured oral mucosal epithelial cells (COE) in vitro, and identify candidate factors that induce corneal neovascularization after transplantation of COE sheets. Rabbit corneal and oral mucosal epithelial cells were co-cultured with mitomycin C-treated NIH/3T3 cells on culture plates and inserts. After CCE and COE were multilayered, culture medium was replaced by basal medium and incubated. Angiogenic potential was examined by invasion, migration and tube formation assays with human umbilical vein endothelial cells (HUVECs). Protein secretion of fibroblast growth factor 2 (FGF2), vascular endothelial growth factor (VEGF), angiopoietin-1 and transforming growth factor beta1 was assessed in conditioned medium by ELISA. Gene expression of FGF2 and VEGF was also quantified by real-time RT-PCR and neutralizing antibodies against FGF2 and VEGF were employed for blocking assays. COE induced significantly greater invasion, migration and tube formation of HUVECs, when compared to CCE. CCE secreted a significantly lower amount of FGF2 than COE, while amounts of VEGF were approximately equal in both culture media. Similarly, significantly higher expression of FGF2 mRNA was observed with COE, while no significant difference in VEGF mRNA expression was observed between COE and CCE. Only anti-FGF2 neutralizing antibody significantly suppressed HUVEC invasion and migration induced by COE, without suppression in CCE. In conclusion, angiogenic potential of COE is greater than that of CCE and FGF2 is a candidate involved in the induction of corneal neovascularization after COE sheet transplantation.
本研究旨在比较培养的角膜上皮细胞（CCE）和培养的口腔黏膜上皮细胞（COE）在体外诱导血管生成的能力，并确定COE片移植后诱导角膜新生血管形成的候选因子。将兔角膜和口腔黏膜上皮细胞与经丝裂霉素C处理的NIH/3T3细胞在培养板和小室中共同培养。在CCE和COE形成多层后，用基础培养基替换培养基并进行孵育。通过用人脐静脉内皮细胞（HUVECs）进行侵袭、迁移和管形成试验来检测血管生成潜力。通过ELISA评估条件培养基中成纤维细胞生长因子2（FGF2）、血管内皮生长因子（VEGF）、血管生成素-1和转化生长因子β1的蛋白分泌。还通过实时RT-PCR定量FGF2和VEGF的基因表达，并使用针对FGF2和VEGF的中和抗体进行阻断试验。与CCE相比，COE显著诱导HUVECs更强的侵袭、迁移和管形成。CCE分泌的FGF2量明显低于COE，而两种培养基中VEGF的量大致相等。同样，COE中FGF2 mRNA的表达明显更高，而COE和CCE之间VEGF mRNA表达没有显著差异。只有抗FGF2中和抗体显著抑制COE诱导的HUVECs侵袭和迁移，而对CCE没有抑制作用。总之，COE的血管生成潜力大于CCE，FGF2是COE片移植后诱导角膜新生血管形成的一个候选因子。
