摘要背景 研究已证实蓝光照射可导致视网膜色素上皮细胞(RPE)凋亡,但其机制目前尚不完全清楚. 目的 探讨线粒体凋亡通路是否参与蓝光照射诱导体外培养的人RPE细胞凋亡过程.方法 分离新鲜的供体视网膜,对人RPE细胞进行原代培养和传代,用角蛋白单克隆抗体行细胞鉴定.将体外培养的人RPE细胞分为无光照组、单纯光照组、光照+硝苯地平组、光照+钙磷酸结合蛋白C(calphostin C)组、光照+佛波酯(PMA)组.光照组细胞用(2 000±500) lx的蓝光照射人RPE细胞6h,然后继续培养24 h后终止.采用Western blot法比较两个组间RPE细胞中凋亡相关调控因子bax、bcl-2、bcl-xl的相对表达,以评价蓝光照射对RPE细胞凋亡的影响.光照+硝苯地平组、光照+calphostin C组、光照+PMA组细胞在蓝光照射前1h分别在培养基中加入相应药物,然后以(2 000±500) lx的蓝光照射人RPE细胞6h,并继续培养24 h,采用Westernblot法检测5个组细胞中caspase-9蛋白表达量的变化,观察钙通道和蛋白激酶C(PKC)通路对RPE细胞线粒体的影响.结果 培养的细胞生长良好,细胞质内充满色素颗粒,呈铺路石样排列,对角蛋白呈阳性反应.无光照组和单纯光照组均可见bax、bcl-2及bcl-xl蛋白条带,相对分子质量分别为23 000、26 000和30 000.与无光照组比较,单纯光照组bax、bcl-2和bcl-xl蛋白表达相对值(A)下降,差异均有统计学意义(t=-4.409,P=0.012;t=7.575,P=0.002;t=6.068,P=0.004).与无光照组比较,单纯光照组、光照+calphostin C组、光照+PMA组细胞中caspase-9蛋白表达均升高,差异均有统计学意义(P=0.005、0.002、0.000),而光照+硝苯地平组与无光照组比较差异无统计学意义(P=0.191).与单纯光照组比较,光照+PMA组caspase-9蛋白表达升高,差异有统计学意义(P=0.005);而光照+硝苯地平组及光照+calphostin C组caspase-9蛋白表达差异均无统计学意义(P=0.057、0.643). 结论 蓝光致体外培养的人RPE细胞凋亡,同时细胞中caspase-9表达增强,凋亡抑制基因bcl-2及bcl-xl表达下降,凋亡促进基因bax蛋白表达增强.线粒体凋亡通路参与蓝光照射致RPE细胞凋亡的过程;PKC通路可能参与了蓝光导致的人RPE细胞凋亡.

abstractsBackground Studies determined that blue light exposure causes apoptosis of human retinal pigment epithelial (RPE) cells,but its mechanism is still below understood.Objective The aim of this study was to investigate whether or how mitochondrial apoptotic pathway is involved in blue-light induced apoptosis of human RPE cells in vitro.Methods Human RPE cells were isolated from fresh donor eyes and primarily cultured and passaged.The cells were identified with keratin antibody by immunochemistry.Then the cells were the non-light exposed group,simple light-exposed group,light-exposed+nifedipine group,light-exposed+calphostin C group and the light-exposed+phorbol myristate acetate (PMA) group.Human RPE cells in light-exposed group were consequently cultured for 24 hours following the exposure of (2 000±500)lx blue-light for 6 hours,and then the expression levels of bax,bcl-2,bcl-xl in the cells were detected by Western blot to evaluate the effect of blue light on the apoptosis.The cells in the light-exposed+nifedipine group,light-exposed+calphostin C group and the light-exposed+PMA group were treated with the corresponding drugs 1 hour prior to light irradiation and sequently received 6-hour light irradiation and 48-hour culture.The expression of caspase-9 protein in the cells were assayed with Western blot to assess the influence of Ca2+ channel and protein kinase C (PKC) pathway on mitochondria of RPE cells.Results Cultured cells grew well with visible pigment in cytoplasm.The cells showed the positive response for keratin and presented a cobblestone-like appearance.The expression bands of bax,bcl-2 and bcl-xl proteins were clearly visible at the molecular weight of 23 000,26 000 and 30 000 in both non-light exposed group and the simple light-exposed group,and the absorbance values of the cells to bax were elevated,while the absorbance values to bcl-2 and bcl-xl were declined in the simple light-exposed group compared with the non-light exposed group (t =-4.409,P =0.012 ;t =7.575,P =0.002 ; t =6.068,P =0.004).Compared with the non-light exposed group,the absorbance values of caspase-9 were significantly raised in the simple light-exposed group,light-exposed+calphostin C group and the lightexposed+PMA group (P=0.005,0.002,0.000),but no significant difference between the non-light exposed group and light-exposed+nifedipine group (P=0.191).Compared with the simple light-exposed group,the expression level was considerably higher in the light-exposed + PMA group (P =0.005) ; while that in the light-exposed + nifedipine group or light-exposed+calphostin C group was not significantly different (P=0.057,0.643).Conclusions Blue light exposure induces apoptosis of RPE cells by up-regulating the expressions of bax and caspase-9 proteins and down-regulating the expressions of bcl-2 and bcl-xl.The mitochondrial apoptosis pathway and PKC pathway participate in blue-light induced apoptosis of human RPE cells in vitro.