Suppression of corneal neovascularization by culture supernatant of human amniotic epithelial cells in vitro / 中华实验眼科杂志

ABSTRACT

BackgroundCorneal neovascularization (CNV) is a common eye disease.The researches on the pathogenesis and treatment of CNV are focus in Ophthalmology.ObjectiveThis study was to investigate the effects of culture supernatant from human amniotic epithelial cells (AECs) on CNV in vitro and its mechanism.MethodsHuman AECs were obtained from a placenta and cultured in serum-free medium for 48 hours,and the supernatant was collected.The levels of interleukin-1 receptor antagonist (IL-1 Ra) and pigment epithelium-derived factor (PEDF) in the human AECs culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).Rabbit corneal epithelial cells (CECs) were obtained and cultured in different concentrations of human AECs culture supernatant for 48 hours,serum-free medium was used as the control group.The expression of vascular endothelial growth factor (VEGF) mRNA and basic fibroblast growth factor (bFGF) mRNA in rabbit CECs were measured by quantitative real-time PCR (QRT-PCR).Human umbilical cord vein endothelial cells (UVECs) were cultured in the three mediums above,and the proliferation of human UVECs (absorbance value,A value) was tested by the cell counting kit 8 ( CCK8 ).Migration assay was performed by the wound healing method for the human UVECs.The membrane ultra-structure of human UVECs was examined under the atomic force microscope (AFM).ResultsCultured and passaged human AECs showed a positive response for keratin.The expression of VEGF mRNA (1.00±0.22) and bFGF mRNA (1.00±0.36) in rabbit CECs was suppressed by the human AECs culture supernatant,with a significant reduction in comparison with the serum-free DMEM group (2.98±0.46,2.55±0.48 )(P=0.001,0.002).The A value was significantly lowered in the human AECs culture group for 72 hours compared with the serum-free DMEM group ( 1.941 ± 0.036 versus 2.144 ± 0.059 ) ( P =0.000 ),and the bFGF-induced migration rate of human UVECs was strongly inhibited by the culture supernatant of human AECs in comparison with serum-free DMEM.The plasma membrane of human UVECs cultured with the human AECs culture supernatant was full of bumps,and decreased intercellular connection and cellular pseudopodia were found on the AFM image.The concentration of IL-1Ra was (153.56±0.36)ng/L and that of PEDF was (70.41 ±0.68 )μ,g/L in the human AECs culture supernatant.Nothing was deteched in serum-free DMEM group.Conclusions Human AECs culture supernatant suppressed the expression of VEGF and bFGF in CECs as well as the migration and proliferation of vascular endothelial cells.The effect may be associated with IL-1Ra and PEDF secreted by human AECs.These results suggest that human AECs may be a potential therapy for the inhibition of CNV.