| Objective:To establish blue-light damage model of RPE, investigate whether L-type calcium channels are involved in human retinal pigment epithelial (RPE) cells injury blue light induced, and the relationships among blue light exposure, calcium channel subtypes in the heart (α1 C or CaV1.2), neuroendocrine subtype (α1D or CaV1.3) and retinal subtypes (α1F or CaV1.4) messenger ribonucleic acid (mRNA) expression and observe concentration change of free calcium in intracellular RPE cells.Methods:The fourth-generation human RPE cells were randomly divided into four groups. The cells were exposed to blue light(2000±5001ux) for 3,6,9,12 hours respectively, cells culture was stopped at 24 hours later. TUNEL assay was adopted to determine the most suitable light time. First, the fourth generation human RPE cells in vitro were randomly divided into four groups:control group (no light group), light group, light+nifedipine group, and light+(-) BayK8644 group. Then, the cells were exposed to blue light(2000±5001ux) for 6 hours,24 hours prolongation of post-exposure culture. Reverse transcription- polymerase chain reaction real time RT-PCR and flourescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype (α1C or CaV1.2), neuroendocrine subtype (α1D or CaV1.3) and retinal subtypes (α1F or CaV1.4) in each group. Then the fourth generation cells were randomly divided into 6 groups:control group (no light group), light group, nifedipine group, light + nifedipine group, (-) BayK8644 group, light+(-) BayK8644 group. The cells were exposed to blue light(2000±5001ux) for 6 hours, cells culture was stopped at 24 hours later. The laser confocal microscopy was used to detect intracellular calcium concentration. The excitation wavelength was 488nm, emission wavelength was 505nm. Each of 30 cells was randomly selected. The cell images were scaned, the analysis software was used to analyze cell fluorescence intensity of intracellular calcium. One-factor analysis of variance was used to analyze intracellular fluorescence intensity differences.Results In 24 hours prolongation of post-exposure culture group, the apoptotic index in blue-light exposure for 6,9,12 hours group was higher than thos of controll and group 3 hours. The cDNA molecular weight ofα1C,α1D, alF subunit and internal parameter of ATCB are 68bp,157bp,125bp and 186bp respectively. Gel electrophoresis showed that the position of products was consistent with above molecular weight. (1)α1C mRNA expression in each group,α1C mRNA expression in light, light + Nifedipine and light+(-) BayK.8644 group was higher than those in control group, the difference was significantly different (P=0.002,0.001,0.000), light +Nifedipine group, light+(-) BayK8644 group were higher than those of light group (P=0.003,0.000), light+(-) BayK8644 group were higher than those of light +Nifedipine group, the difference was significantly different (P=0.004). (2)α1D mRNA expression in each group, alD mRNA expression in light, light+Nifedipine and light+(-) BayK8644 group was higher than those in control group, the difference was statistical significance (P=0.023,0.006,0.010), light+(-) BayK8644 group was higher than those of light group and light+Nifedipine group (P=0.032, P=0.039), light group and the light+Nifedipine group were not statistical significance (P=0.459).(3)α1F mRNA expression in each group,α1F mRNA expression in light, light+ Nifedipine and light+(-) BayK.8644 group was higher than those in control group, there was statistical significance (P=0.010,0.000,0.002), light +Nifedipine group and light+(-) BayK8644 group were higher than those of light group (P=0.000, 0.005), light+Nifedipine group and the light+(-) BayK8644 group was not statistical significance (P=0.585).Confocal laser scanning microscope showed that fluorescence intensity of intracellular calcium 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); The intracellular calcium fluorescence intensity in Nifedipine group was lower than that of control and light +Nifedipine group, there were statistical significance (P=0.000,0.000). The difference between Nifedipine group and light + Nifedipine were significantly (P=0.000), The difference between light + Nifedipine group and light group, (-) BayK8644 group and the light+(-) BayK.8644 group, were not significantly different (P=0.204,0.103).Conclusion (1) The best conditions to establish blue light damage model of human retinal pigment epithelial cells in vitro are 2000±500Lux light intensity,6 hours time, 24 hours prolongation of post-exposure culture. (2) The blue light exposure can cause L-type calcium channel subunit ofαlC,α1D andα1F mRNA expression to increase differently degree. Application of the 10-5M Nifedipine could not be resistant affection blue light-reduced onα1D mRNA expression, while application of the Ca2+ channel activating (-) BayK.8644 is opposite. L-type calcium channel may be involved in blue light induced damage on human retinal pigment epithelial cells. (3) Blue light exposure over threshold can induce damage to human RPE cells, probably by triggering the increase of intracellular free calcium concentration. (4) The affections of blue light on L-type calcium channel in the human RPE cells were probably stronger than 10-5M Nifedipine. |
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