蓝光照射对人视网膜色素上皮细胞钙离子-蛋白激酶C信号通路的影响

The effect of blue light on Ca2＋-protein kinase C signaling pathway in human retinal pigment epithelial cells in vitro

目的 观察蓝光照射对人视网膜色素上皮细胞钙离子（Ca2-）-蛋白激酶C（PKC）信号通路的影响.方法 体外培养并鉴定人视网膜色素上皮（RPE）细胞,取第4代人RPE细胞随机分组进行实验.采用蛋白免疫印迹法测定培养细胞PKC蛋白表达,检测佛波酯（PMA）和钙磷酸结合蛋白（calphostin C）对PKC活性的影响,确定PMA与calphostin C影响PKC活性的最适宜浓度.采用非放射性核素法测定蓝光照射处理对培养细胞PKC活性的影响.采用20 W,波长450～500 nm医用蓝光灯作为光源,光照强度（2000±500） Lux,照射6h,24 h后终止培养,以此制造体外培养人RPE细胞光损伤.将培养细胞随机分成5个组,即无光照、单纯光照、光照联合硝苯地平、光照联合calphostin C、光照联合PMA组.其中,无光照组不接受光照;单纯光照组仅接受光照;光照联合硝苯地平组接受光照和0.1 mmol/L的硝苯地平;光照联合calphostin C组接受光照和100.0 nmol/L的calphostin C;光照联合PMA组接受光照和100.0 nmol/L的PMA.用乙酰氧基甲基酯Ca2＋荧光探针标记各组培养细胞,激光扫描共焦显微镜测定各组细胞内Ca2＋浓度.比较各组细胞内Ca2＋浓度差异.结果 经鉴定,体外培养人RPE细胞成功.100.0、200.0 nmol/L PMA处理的RPE细胞中PKC蛋白相对表达量高于0.1、1.0、10.0、50.0 nmol/L PMA处理的RPE细胞,差异有统计学意义（F=217.537,P＜0.05）,但100.0、200.0 nmol/L PMA处理组间PKC蛋白相对表达量比较,差异无统计学意义（P=0.072）.100.0、200.0 nmol/L calphostin C处理的RPE细胞中PKC蛋白相对表达量低于5.0、25.0、50.0、75.0 nmol/L calphostin C处理的RPE细胞,差异有统计学意义（F=164.543,P＜0.05）,但100.0、200.0 nmol/L calphostin C处理组间PKC蛋白相对表达量比较,差异无统计学意义（P=0.385）.蓝光照射处理后,RPE细胞的PKC活性显著升高,与未接受蓝光照射处理的RPE细胞的PKC活性比较,差异有统计学意义（t=-9.869,P＜0.05）.单纯光照、光照联合硝苯地平、光照联合Calphostin C、光照联合PMA组的RPE细胞内Ca2＋浓度均高于无光照组,差异有统计学意义（F=26 764.92,P＜0.05）;光照联合PMA组RPE细胞内Ca2＋浓度高于单纯光照、光照联合硝苯地平及光照联合calphostin C组（P＜0.05）,单纯光照组高于光照联合硝苯地平和光照联合calphostin C组（P＜0.05）.结论 蓝光照射后人RPE细胞内PKC活性增高,Ca2＋浓度增高.硝苯地平和calphostin C均能降低蓝光照射后人RPE细胞内Ca2＋浓度,PMA增加细胞内Ca2＋浓度.

Objective To investigate the effect of blue light on Ca2＋-protein kinase C （PKC）signaling pathway in human retinal pigment epithelial （RPE） cells in vitro.Methods Primary human RPE cells were cultured in vitro and characterized.The experiments were carried out using the 4th generation of human RPE cells.The PKC protein level was measured by Western blot to determine the most appropriate concentration of phorbol ester （PMA） and calcium phosphate binding protein （calphostin C） on PKC expression.Non-radioactive isotope method was used to determine the effect of blue light on PKC expression of cultured cells.Blue-light damage model of human RPE cells was established by 6 hour irradiation of medical blue-light lamp [20 W,450-500 nm wavelength,（2000＆#177; 500） Lux],and 24 hours prolongation of post-exposure culture.The human RPE cells were randomly divided into 5 groups.Group A did not receive light irradiation,group B only received blue light irradiation,group C was blue light irradiation and 0.1 mmol/L nifedipine treatment,group D was blue light irradiation and 100.0 nmol/L calphostin C treatment,group E was blue light irradiation and 100.0 nmol/L PMA treatment.Intracellular Ca2-concentration was measured by acetoxymethyl ester （Fluo 3-AM） labelling and confocal microscope imaging.Results The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L PMA-treated groups was higher than 0.1,1.0,10.0,and 50.0 nmol/L PMA-treated groups,the difference was statistically significant （F=217.537,P〈0.05）,but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L PMA-treated groups （P =0.072）.The PKC protein expression in 100.0 nmol/L or 200.0 nmol/L calphostin C-treated groups was lower than 5.0,25.0,50.0,and 75.0 nmol/L calphostin C-treated groups,the difference was statistically significant （F=164.543,P〈0.05）,but there was no statistically difference between 100.0 nmol/L and 200.0 nmol/L calphostin C treated groups （P=0.385）.PKC level in blue light group was higher than non-light group,the difference was statistically significant （t =-9.869,P〈0.05）.The Ca2＋ fluorescence intensity values in group B,C,D and E was higher than group A,the difference was statistically significant （F=26 764.92,P〈0.05）.The Ca2＋ fluorescence intensity values in group E was higher than group B,C and D （P〈0.05）,and that in group B was higher than group C and D （P〈0.05）.Conclusions The PKC activity and intracellular Ca2＋ concentration in human RPE cells increase after blue light irradiation.Both calcium channel inhibitor nifedipine and PKC inhibitor calphostin C can reduce intracellular Ca2＋ concentration in human RPE cells.PMA can induce intracellular Ca2＋ concentration in human RPE cells after blue light irradiation.