背景 细胞质膜钙ATP酶3(PMCA3)参与维持晶状体上皮细胞(LECs) Ca2+的平衡,可能与白内障的病理过程有关,紫外线B(UVB)是引起白内障的重要因素之一,但UVB对LECs中PMCA3表达的影响少有报道. 目的 研究UVB对人LECs系B-3(HLE B-3) PMCA3表达的影响. 方法 对HLE B-3细胞进行培养和传代,当细胞达80％以上融合时分别暴露于用不同剂量(0、5、10、20 mJ·s/cm2)的UVB分别照射0、20、40和80 s,然后继续培养24、48和72 h,用MTT法检测不同剂量UVB照射后细胞的生存率;用JC-1染色法检测UVB照射后细胞线粒体膜电位(△ψm)的变化;以DCFH-DA染色法检测UVB照射后细胞内活性氧簇(ROS)的变化;采用annexin V-FITC/PI染色法检测UVB照射后细胞的凋亡情况;用Fluo-3/AM染色法检测细胞内Ca2+浓度的变化;分别采用实时荧光定量PCR(real-time PCR)法和Western blot法检测UVB照射后细胞中PMCA3 mRNA及PMCA蛋白的表达变化. 结果 随着UVB照射剂量的增加和照射时间的延长,细胞生存率均明显下降,差异均有统计学意义(F分组=72.411,P=0.000; F时间=36.588,P=0.000),其中10 mJ·s/cm2、20 mJ·s/cm2 UVB照射后24 h HLE B-3细胞的生存率分别为(75.3±2.2)％和(48.7±4.5)％,明显低于对照组的(100.0±0.0)％,差异均有统计学意义(P=0.001、0.000);5、10、20 mJ·s/cm2 UVB照射后48 h细胞的生存率分别为(84.9±1.2)％、(69.3±17.4)％和(32.8±4.5)％,均明显低于对照组的(100.0±0.0)％,差异均有统计学意义(P=0.047、0.000、0.000);5、10、20 mJ·s/cm2 UVB照射后72 h细胞的生存率分别为(55.1±3.0)％、(42.1±1.9)％和(26.1±4.7)％,与对照组的(100.0±0.0)％相比生存率明显降低,差异均有统计学意义(均P=0.000).JC-1染色法检测表明,对照组细胞内可见红色荧光,5mJ·s/cm2 UVB照射后细胞内出现绿色荧光,10 mJ·s/cm2及20 mJ·s/cm2 UVB照射组出现绿色荧光增强,红色荧光减弱.5、10、20 mJ· s/cm2 UVB照射后细胞内的ROS由0.4％分别增加至35.8％、51.9％和76.7％.0 mJ·s/cm2 UVB照射组凋亡和坏死细胞比例为2.0％,5、10、20 mJ·s/cm2 UVB照射组凋亡和坏死细胞率分别为4.2％、7.6％和15.1％.10 mJ·s/cm2和20 mJ·s/cm2 UVB照射组细胞内Ca2+水平分别为0 mJ·s/cm2照射组的(1.2±0.1)倍和(1.3±0.1)倍,差异均有统计学意义(P=0.039、0.004).5、10和20 mJ·s/cm2 UVB照射组HLE B-3细胞PMCA3 mRNA表达量均明显低于对照组,差异均有统计学意义(P=0.001、0.004、0.000),各组细胞中的PMCA蛋白相对表达量也均明显低于对照组,差异均有统计学意义(P=0.000、0.000、0.001). 结论 UVB照射可导致白内障发生的机制可能与人LECs中PMCA3表达量下降和诱导LECs凋亡有关,UVB的作用呈剂量和时间依赖性.%Background Plasma membrane calcium ATPase 3 (PMCA3) participates in the regulation of Ca2+ level in lens epithelial cells (LECs),which may be associated with the pathogenesis of cataract.It has been proved that ultraviolet B (UVB) is one of causing-factors of cataract.However,the effect of UVB on the expression of PMCA3 in LECs is unclear.Objective This study was to investigate the effects of UVB irradiation on the expression of PMCA3 in human LECs B-3.Methods HLE B-3 cells were cultured and passaged.The cells were exposed to 0,5,10 and 20 mJ · s/cm2 UVB for 0,20,40,80 s,respectively and further cultured for 24,48 and 72 hours.MTT assay was used to detect the cell proliferation rate.JC-1 staining was used to detect the changes of mitochondrial membrane potential (△ψm) in the cells.The intracellular reactive oxygen species (ROS) level was detected by DCFH-DA staining,and cell apoptosis was evaluated using annexin V-FITC/PI staining.In addition,the intracellular calcium ion (Ca2+) concentration in the cells was assayed with Fluo-3/AM staining.The expression levels of PMCA3 mRNA and PMCA protein in the HLE B-3 cells were detected by real-time PCR and Western blot,respectively.Results The survival rates of the cells were significantly reduced with the increase of irradiated intensity of UVB and the lapse of time (Fgroup =72.411,P =0.000 ; Ftime =36.588,P =0.000),and the survival rates of the cells in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB for 24 hours were (75.3 ± 2.2) ％ and (48.7 ±4.5) ％,respectively,which were significantly lower than those in the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P=0.001,0.000).The survival rates of the cells in the 5,10,20 mJ · s/cm2 UVB for 48 hours were (84.9± 1.2) ％,(69.3±17.4)％ and (32.8±4.5)％,showing significant declines in comparison with the 0 mJ · s/cm2 UVB group ([100.0±0.0] ％) (P =0.047,0.000,0.000).In 72 hours following 5,10,20 mJ · s/cm2 UVB irradiation,the survival rates of the cells were (55.1 ± 3.0) ％,(42.1 ± 1.9) ％ and (26.1 ±4.7) ％,respectively,with significant differences in comparison with the 0 mJ · s/cm2 UVB group ([100.0 ± 0.0] ％) (P =0.000,0.000,0.000).JC-1staining exhibited the intracellular red fluorescence in the normal cells group.However,in the 5 mJ · s/cm2 UVB group,weak green fluorescence was seen,and the green fluorescence was enhanced in the 10 mJ · s/cm2 and 20 mJ · s/cm2 UVB groups.After irradiated by 5,10 and 20 mJ · s/cm2 UVB,the ROS levels in the cells increased from 0.4％ to 35.8％,51.9％ and 76.7％,respectively.The apoptosis and necrosis rate of the cells was 2.0％ in the 0 mJ · s/cm2 UVB and 4.2％,7.6％,15.1％ in the 5,10,20 mJ · s/cm2 UVB groups,respectively.The Ca2+ level raised by (1.2±0.1) and (1.3±0.1) folds in the 10 and 20 mJ · s/cm2 UVB groups more than that in the 0 mJ · s/cm2 group (P =0.039,0.004).The expression levels of PMCA3 mRNA in the cells were significantly reduced (P=0.001,0.004,0.000),and the expression levels of the PMCA protein were declined in the 5,10 and 20 mJ · s/cm2 UVB groups compared with the 0 mJ · s/cm2 UVB group (P=0.000,0.000,0.001).Conclusions UVB irradiation causes cataract probably through downregulating the expression of PMCA3 in human LECs and inducing apoptosis of LECs in a dose-and time-dependent manner.
展开▼
