Suppression Of Corneal Neovascularization By Culture Supernatant Of Human Amniotic Epithelial Cells In Vitro

Object This study is to investigate the effects of supernatant of cultured humanamniotic epithelial cells (HAECs) on corneal neovascularization in vitro.Methods HAECs were obtained from a placenta and cultured in serum-free mediumfor48hs, and the supernatant was collected. The level of pigment epithelium-derivedfactor (PEDF) and Interleukin-1receptor antagonist (IL-1Ra) in HAECs culturesupernatant were detected by enzyme-linked immunosorbent assay (ELISA) assay.Rabbit corneal epithelial cells (RCECs) were obtained and cultured in differentconcentrations of HAECs culture supernatant for48hs, serum-free medium was usedas a control. The expression of vascular endothelial growth factor (VEGF) and basicfibroblast growth factor (bFGF) and Interleukin-1beta (IL-1β) in RCECs wereconfirmed at the RNA level by Real-time quantitative PCR (QRT-PCR). Theproliferation of RCECs was tested by CCK8assay. The proliferation of humanumbilical cord vein endothelial cells (HUVECs) was tested by CCK8assay. Migrationassay was carried out by wound healing method. The membrane ultra-structure ofHUVECs was analyzed by atomic force microscope (AFM).Result Cultured HAECs showed the positive response for keratin. Concentrations of(70.41±0.68) μg/L of PEDF and (153.56±0.36) ng/L of IL-1Ra were detected inHAEC culture supernatant. The expressions of VEGF mRNA (1.00±0.22) and bFGFmRNA(1.00±0.36) and IL-1βmRNA(1.00±0.02) in rabbit corneal epithelial cells weresuppressed by HAECs culture supernatant with the significant reduced in comparisonwith serum-free DMEM group (2.98±0.46,2.55±0.48,32.88±0.11)(P<0.05). Theproliferation of RCECs was enhanced by culture supernatant of HAECs (2.321±0.034)compared with serum-free DMEM group (1.862±0.039). The proliferation ofHUVECs was significantly lowed in HAECs culture group (1.941±0.036) comparedwith serum-free DMEM group (2.144±0.059).The migration of HUVECs werestrongly inhibited by culture supernatant of HAECs culture supernatant comparedwith control. The membrane of HUVECs cultured by HAECs culture supernatant was full of bump and was more roughness compared with control according to AFMimages.Conclusion HAECs culture supernatant inhibits corneal neovascularization in vitro.This effect may be elicited in part through the inhibiting expression of VEGF andbFGF and IL-1β of corneal epithelial cells, and partly by inhibition of migration andproliferation of vascular endothelial cells. The effect may be associated with IL-1Raand PEDF secreted by HAECs.