蓝光对人视网膜色素上皮细胞α1D亚基蛋白表达及分泌VEGF和bFGF的影响

The effect of blue light on human retinal pigment epithelium cells α1 D subunit protein expression and vascular endothelial growth factor and basic fibroblast growth factor secretion in vitro

目的 探讨蓝光对人RPE细胞α1D亚基蛋白表达及分泌VEGF和bFGF的影响.方法 实验研究.培养的人RPE细胞分为对照组;光照组;光照＋硝苯地平组;光照＋（-）BayK8644组.分别用（2 000 ±500）lx蓝光照射6h,终止细胞培养24 h.采用酶联免疫法测定RPE细胞分泌的VEGF和bFGF;Real Time-PCR检测各组RPE细胞L型钙通道中神经内分泌亚型（α1D或CaV1.3） mRNA的表达;免疫印迹（Western blot）法检测α1D亚基的蛋白表达.各组VEGF和bFGF浓度,α1D亚基mRNA及蛋白表达比较采用方差分析,两两比较采用LSD法.α1D亚基蛋白表达量与分泌VEGF及bFGF浓度之间的关系采用相关性分析.结果 （1）4组VEGF和bFGF浓度的总体均数比较有统计学意义（F=99.441、21.310,P=0.000、0.000）.光照组（3 281.51±251.73、1 346.81±62.27）与光照＋（-）Bay K8644组（3 808.01±94.01、1 485.82±108.97） VEGF、bFGF浓度高于对照组（2 401.09±228.07、1 232.42±65.41）,差异有统计学意义（P=0.000,0.000,0.019,0.000）;光照＋（-）Bay K8644组（3 808.01±94.01、1 485.82±108.97）高于光照组（3 281.51±251.73、1 346.81±62.27） （P=0.000,0.006）;光照＋硝苯地平组（1 927.28±143.11、1 149.39±62.99）低于光照组（3 281.51±251.73、1 346.81±62.27） （P=0.000,0.000）.（2）4组α1D亚基mRNA表达总体均数比较有统计学意义（F=50.320,P=0.000）.光照组（210 ±23）、光照＋硝苯地平组（232±14）、光照＋（-）BayK8644组（478±80）α1D mRNA的表达均高于无光照组（100±20）,差异均有统计学意义（P =0.023、0.006、0.010）;光照＋（-）BayK8644组（478±80）高于光照组（210±23）及光照＋硝苯地平组（232±14） （P =0.032、0.039）.（3）4组α1D亚基蛋白表达总体均数比较有统计学意义（F=1 940.363,P=0.000）.光照组（0.974 2±0.014 7）、光照＋（-）Bay K8644组（0.654 9±0.005 0）α1D亚基/βactin吸光度值均高于对照组（0.503 2±0.007 5）,差异有统计学意义（P=0.000,0.000）;光照＋（-）Bay K8644组（0.654 9±0.005 0）高于光照＋硝苯地平组（0.413 9 ±0.008 8）（P =0.000）.（4）蓝光照射后α1D亚基蛋白表达量与分泌VEGF浓度之间存在正相关（r =0.674,F=8.333,P=0.016）,与分泌bFGF浓度之间无相关性（r=0.537,F=4.061,P=0.072）.结论 L型钙通道α1D亚基参与了蓝光致RPE细胞的损伤过程,蓝光照射导致人RPE细胞α1D亚基mRNA及蛋白表达量升高,VEGF和bFGF分泌量增加,α1D亚基蛋白表达与分泌VEGF量之间呈正相关.

Objective To investigate the effect of blue light on human retinal pigment epithelium （RPE） α1 D subunit protein expression and its relationship with vascular endothelial growth factor （VEGF）and basic fibroblast growth factor （bFGF） secretion in vitro.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 （2 000 ± 500） lx for 6 hours,and cells culture completed 24 hours later.VEGF and bFGF concentration were assayed by enzyme linked immunosorbent assay （ELISA）.Realtime polymerase chain reaction was used to analysis L-type calcium channel α1 D subunit mRNA expression.Western blot was used to examine the protein expression of L-type calcium channel α1D subunit.Analysis of variance was used to compare the difference of α1 D subunit mRNA and protein expression,VEGF and bFGF concentration between groups.Correlation analysis was used to show the relationship between α1 D subunit protein expression and concentration of VEGF and bFGF.Results （1） There is significant statistically difference in the population mean of VEGF and bFGF concentration in four groups （F =99.441,21.310,P =0.000,0.000）.VEGF and bFGF concentration in group B（3 281.51 ±251.73,1 346.81 ±62.27） and group D （3 808.01 ± 94.01,1 485.82 ± 108.97）was higher than it was in group A （2 401.09 ± 228.07,1 232.42 ±65.41）,which was statistically different （P =0.000,0.000,0.019,0.000）.And it was higher in group D（3 808.01 ±94.01,1 485.82 ± 108.97） compared with group B（3 281.51 ±251.73,1 346.81 ± 62.27） （P =0.000,0.006）.While,it was lower in group C （1 927.28 ± 143.11,1 149.39 ± 62.99） than it was in group B （3 281.51 ± 251.73,1 346.81 ± 62.27） （P =0.000,0.000）.（2） The mean of mRNA expression of α1D subunit between four groups was statistically significant （F =50.320,P =0.000）.It was higher in group B（210 ±23）,group D （478 ±80） and group C（232 ± 14） than group A （100±20）.It was statistically significant different （P =0.023,0.006,0.010）.And it was higher in group D（478 ± 80） than group B（210±23） and C（232±14） （P=0.032,0.039）.（3） There was statistically significant difference in the expression of L-type calcium channel α1D subunit protein in four groups （F =1 940.363,P =0.000）.It was significantly higher （P =0.000,0.000） in group B （0.974 2 ± 0.014 7）and group D（0.654 9 ±0.005 0）than group A （0.503 2 ±0.007 5）.And it was higher in group D （0.654 9 ± 0.005 0） than it was in group C （0.413 9 ± 0.008 8） （P =0.000）.（4） There was positive correlation between the L-type calcium channel α1D subunit protein expression and VEGF concentration secreted by retinal pigment epithelium cells（r =0.674,F =8.333,P =0.016）,but there was no correlation with bFGF concentration （r =0.537,F =4.061,P =0.072）.Conclusions L-type calcium channel α1 D subunit may be involved in blue light induced damage on human retinal pigment epithelial cells.Blue light exposure can induce the mRNA and protein expression of α1 D subunit,VEGF and bFGF concentration in retinal pigment epithelium cells Increased.And there was a positive correlation between α1 D subunit protein expression and the VEGF concentration.