The Effect Of Blue Light On α1D Subunit Protein Expression And Secretion Function Of Human Retinal Pigment Epithelium Cells In Vitro

Objective:To investigate the effect of blue light on intra-cellular calciumion concentration and secretion vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and pigment epithelium-derived factor (PEDF) of cultured human retinal pigment epithelium (RPE) cells in vitro, to detect the relation between the protein expression of L-type calcium channelα1D subunit and VEGF,bFGF and PEDF secretion change, and whether Dihydropyridines(DHPs) nifedipine and (-) Bay K8644 be interfereing with the results.Methods:The fourth generation cultured human RPE cells in vitro were randomly divided into 4 groups, group A(control), group B(exposure to blue light), group C(exposure to blue light+nifedipine), group D(exposure to blue light+(-) Bay K8644). Cells were exposed to blue light (2000±500lux) for 6 hous, cells culture was stoped 24 hous later. The VEGF, bFGF and PEDF concentration was assayed by enzyme linked immunosorbent assay (ELISA). Western blot was used to examine protein expression of L-type calcium channelα1D subunit protein. Reversetranscription-polymerase chain reaction (RT-RCR) and fluorescence quantitative PCR were used to analysis of L-type calcium channel subunit alD mRNA expression. Laser scanning confocal microscopy was used to detect intracellular calcium concentration.Result(1) Detection of VEGF, bFGF and PEDF concentrationComparing with group A, the VEGF concentration in Group B and D was higher (P=0.000, 0.000), group C was much lower than group B(P=0.000), group D was higher than group B (P=0.000). Comparing with group A, the bFGF concentrations in Group B and D was higher(P=0.000,0.000), group C was lower than group B (P=0.019), group D was much higher than group B (P=0.006). Comparing group A with Group B,C and D, the PEDF concentration difference was no significant difference (P=0.318,0.411,0.860), comparing group B with Group C and D, the difference was no significant difference (P=0.856, 0.243), comparing group C with Group D, the difference was no significant difference(P= 0.321).(2)Protein expression of L-type calcium channelα1D subunitProtein expression of L-type calcium channelα1D subunit was significantly higher in group B and D than group A (P=0.000,0.000). Group C was lower than group A (P =0.000). Group D was much higher than Group C (P=0.000). Polyacrylamide gel electrophoresis showed protein strap in 250Kd of molecular weight.(3) The correlation analysis among the protein expression of L-type calcium channel a1D subunit, VEGF and bFGF concentrationProtein expression of L-type calcium channel alD subunit was positive correlation with VEGF secreted retinal pigment epithelium cells (r=0.674, F=8.333, P=0.016).Protein expression of L-type calcium channelα1D subunit was not correlated with bFGF concentration (r=0.537, F=4.061, P=0.072)(4)The intracellular calciumions concentrationThe calciumions concentration in light, (-)Bayk8644 and light+(-)Bayk8644 group was higher than those of control group, the difference was statistical significance (P=0.000, 0.000,0.000). Nifedipine group was lower than control group (P=0.000), light + nifedipine group compared with the light group, light+nifedipine group and the nifedipine group, the difference was statistically significant (P= 0.000,0.000), (-) BayK8644 group and the light+(-) BayK8644 group, the difference was no significant difference (P= 0.155).(5)The mRNA expression ofα1D subunitThe mRNA expression ofα1D subunit in light, light+(-) Bay K8644 and light+ nifedipine were higher than those in control group, the difference was statistically significant (P=0.023,0.006,0.010), light+(-) Bay K8644 group was higher than light and light+nifedipine group (P=0.032,0.039). Compared with the light group, the mRNA expression ofα1D subunit in light+nifedipine group was no significant (P=0.459). Conclusion:(1) Blue light exposure can induce high mRNA and protein expression ofα1D subunit, high VEGF and bFGF concentration, the high protein expression was positive correlation with the VEGF, whereas was not with bFGF concentration. (2) Application of the Ca2+ channel inhibitor nifedipine can reduce the expression of L-type calcium channelα1D subunit and decrease VEGF and bFGF concentration, while application of the Ca2+ channel activator (-) BayK8644 was completely opposite. (3) The calciumion take part in the procedure of blue light induced damage to human retinal pigment epithelium cells in vitro.