关键词: 血管内皮生长因子, 视网膜色素上皮细胞, 三维培养

Abstract:

Objective To investigate the effect of vascular endothelial growth factor (VEGF) expression induced by cyclic stretch and the relationship between VEGF and cyclic stretch in retinal pigment epithelial (RPE) cells. Design Experimental study. Participates ARPE-19 cell line.Methods Flexcell-5000 strain system was applied in 3D-RPE models. According to different stretch elongations, ARPE-19 cell lines were divided into a control group (non-stretch group, Control), A group (20% elongation), B group (10% elongation) and C group (5% elongation). Collecting different time points samples from each group were estimated by a series of tests. Real-time PCR was utilized to detect nucleic acid levels in discrete time points (0, 24, 48 h). VEGFA-165 was examined by Western blot in different time points (0, 24, 48 h). Meanwhile, ELISA was utilize to measure VEGF-A expression in each group of cell supernatant (0, 12, 24, 36 and 48 h). Besides, in order to quantity angiogenesis ability of VEGF in vitro, tube formation assay was performed by time (24、48h) in the human umbilical vein endothelial cells (HUVEC). Main Outcome Measures The expression of VEGFA mRNA, VEGFA-165, and VEGFA expression in cell supernatant, and the tube fomation counts of HVEC. Results Compared with the control group, the expression of VEGFA mRNA in group A, B, and C at 24 h (F=7.99, P=0.009) and 48 h (F=75.09, P=0.000) was significantly higher than that of the control group, and group B VEGFA mRNA expression at any time point was higher than that in group A and C (all P<0.05). The expression of VEGFA-165 protein in the three stretch groups was significantly higher than that in the control group at 24 hours (F=51.62, P=0.000) and 48 hours (F=91.69, P=0.000). Besides, the relative expression level of VEGFA-165 protein in group B at 24 hours（0.794±0.045）was 1.3 fold (P=0.012) in group A and 1.2 fold in group C (P=0.043). The relative expression level of VEGFA-165 protein in group B at 48 h (1.192±0.042) was 1.4 fold that of group A (P=0.0001) and 1.3 fold that of group C (P=0.0001). With the extension of time, the VEGFA concentration in the supernatant of group A, B and C was dramatically higher than that of the control group at 24 h (F=131.16, P=0.0001), 36 h (F=66.56, P=0.0001) and 48h (F=605.19, P=0.0001). In the tube formation experiment, the number of each group meshes was significantly higher than that of the control group at 48 h (F=13.13, P=0.002), but only the group B meshes number were significantly increased at 24 h (P=0.029). Conclusion Cyclic stretch can up regulate the VEGF expression in RPE cells and have the function of angiogenesis in vitro.

Key words: vascular endothelial growth factor, retinal pigment epithelium cell, 3D-culture