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目的:(1)探讨蓝光照射致体外培养人视网膜色素上皮(retinal pigment epithelium,RPE)细胞分泌血管内皮细胞生长因子(vascular endothelial cell growth factor,VEGF)、色素上皮衍生因子(pigment epithelium-derived factor,PEDF)、RPE细胞内三磷酸肌醇(inositol triphosphate,IP3)和二酰甘油(diacylglycerol,DAG)的浓度变化,及其与Ca2+-PKC信号通路之间的关系。(2)进一步探讨Ca2+-PKC信号通路在蓝光照射致人RPE细胞凋亡事件中的作用。
方法:(1)光照条件的确定:用(2000±500)Lux蓝光照射体外培养人RPE细胞6h,终止培养24h。(2)采用酶联免疫分析法(enzyme-linked immunosorbentasssy,ELISA)测定各组细胞分泌VEGF、PEDF、RPE细胞内IP3和DAG的浓度,将体外培养的第四代人RPE细胞随机分为5组,A组:无光照组;B组:蓝光光照组;C组:蓝光光照+PMA组;D组:蓝光光照+Calphostin C组;E组:蓝光光照+硝苯地平组,(3)采用流式细胞仪检测各组细胞的凋亡率,将体外培养的第四代人RPE细胞随机分为3组,A组:无光照组;B组:蓝光光照组;C组:蓝光光照+Calphostin C组+硝苯地平组。
结果:
(1)各组人RPE细胞分泌VEGF和PEDF的变化及VEGF与PEDF的比值
VEGF:蓝光光照组,蓝光光照+PMA组,蓝光光照+硝苯地平组均高于无光照组,差异有统计学意义(P=0.002,0.002,0.016),蓝光光照+Calphostin C组与无光照组比较,差异无统计学意义(P=0.071);蓝光光照+Calphostin C组,蓝光光照+硝苯地平组低于蓝光光照组(P=0.001,0.002);蓝光光照+PMA组与蓝光光照组比较,差异无统计学意义(P=0.999);
PEDF:蓝光光照组,蓝光+PMA组均低于无光照组,差异有统计学意义(P=0.004,0.011);蓝光光照+Calphostin C组,蓝光光照+硝苯地平组高于无光照组(P=0.000,0.000);蓝光光照+Calphostin C组,蓝光光照+硝苯地平组高于蓝光光照组(P=0.000,0.000),蓝光光照+PMA组与蓝光光照组比较,差异无统计学意义(P=0.569);
VEGF与PEDF的比值:蓝光光照组,蓝光光照+PMA组均高于无光照组,差异有统计学意义(P=0.008,0.027),蓝光光照+Calphostin C组,蓝光光照+硝苯地平组与无光照组比较,差异无统计学意义(P=0.361,0.149);蓝光光照+PMA组与蓝光光照组比较,差异无统计学意义(P=0.917);蓝光光照+Calphostin C组,蓝光光照+硝苯地平组低于蓝光光照组,差异有统计学意义(P=0.016,0.015)。
(2)各组人RPE细胞内IP3和DAG的浓度变化
IP3:蓝光光照组,蓝光光照+PMA组,蓝光光照+Calphostin C组,蓝光光照+硝苯地平组均高于无光照组,差异有统计学意义(P=0.003,0.007,0.000,0.000);蓝光光照+PMA组,蓝光光照+Calphostin C组,蓝光光照+硝苯地平组均高于蓝光光照组,差异有统计学意义(P=0.011,0.000,0.000);
DAG:蓝光光照组,蓝光光照+PMA组,蓝光光照+Calphostin C组,蓝光光照+硝苯地平(Nifedipine)组均高于无光照组,差异有统计学意义(P=0.000,0.000,0.000,0.000);蓝光光照+PMA组,蓝光光照+Calphostin C组低于蓝光光照组,差异有统计学意义(P=0.021,0.007)。蓝光光照+硝苯地平组高于蓝光光照组,差异有统计学意义(P=0.000)。
(3)各组人RPE细胞的凋亡率
蓝光光照组,蓝光光照+ Calphostin C+硝苯地平组的人RPE细胞的凋亡率均高于无光照组,差异有统计学意义(P=0.001,0.009);蓝光光照+硝苯地平+Calphostin C组的人RPE细胞的凋亡率低于于蓝光光照组,差异有统计学意义(P=0.038)。
结论:(1)蓝光照射可致人RPE细胞VEGF分泌量增加,PEDF分泌量减少,同时VEGF与PEDF的比值升高,IP3和DAG浓度增加;(2)L型钙通道及Ca2+-PKC信号通路参与调节蓝光照射导致体外培养的人RPE细胞VEGF,PEDF,IP3和DAG浓度变化,可能存在反馈调节机制。(3) Calphostin C与硝苯地平联合应用可抑制蓝光照射导致体外培养的人RPE细胞凋亡。