中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2014年
6期
485-491
,共7页
紫外线B%钙稳态%质膜钙ATP酶3%细胞凋亡%人晶状体上皮细胞
紫外線B%鈣穩態%質膜鈣ATP酶3%細胞凋亡%人晶狀體上皮細胞
자외선B%개은태%질막개ATP매3%세포조망%인정상체상피세포
Ultraviolet B%Calcium homeostasis%Plasma membrane calcium ATPase 3%Apoptosis%Human lens epithelial cell
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.
揹景 細胞質膜鈣ATP酶3(PMCA3)參與維持晶狀體上皮細胞(LECs) Ca2+的平衡,可能與白內障的病理過程有關,紫外線B(UVB)是引起白內障的重要因素之一,但UVB對LECs中PMCA3錶達的影響少有報道. 目的 研究UVB對人LECs繫B-3(HLE B-3) PMCA3錶達的影響. 方法 對HLE B-3細胞進行培養和傳代,噹細胞達80％以上融閤時分彆暴露于用不同劑量(0、5、10、20 mJ·s/cm2)的UVB分彆照射0、20、40和80 s,然後繼續培養24、48和72 h,用MTT法檢測不同劑量UVB照射後細胞的生存率;用JC-1染色法檢測UVB照射後細胞線粒體膜電位(△ψm)的變化;以DCFH-DA染色法檢測UVB照射後細胞內活性氧簇(ROS)的變化;採用annexin V-FITC/PI染色法檢測UVB照射後細胞的凋亡情況;用Fluo-3/AM染色法檢測細胞內Ca2+濃度的變化;分彆採用實時熒光定量PCR(real-time PCR)法和Western blot法檢測UVB照射後細胞中PMCA3 mRNA及PMCA蛋白的錶達變化. 結果 隨著UVB照射劑量的增加和照射時間的延長,細胞生存率均明顯下降,差異均有統計學意義(F分組=72.411,P=0.000; F時間=36.588,P=0.000),其中10 mJ·s/cm2、20 mJ·s/cm2 UVB照射後24 h HLE B-3細胞的生存率分彆為(75.3±2.2)％和(48.7±4.5)％,明顯低于對照組的(100.0±0.0)％,差異均有統計學意義(P=0.001、0.000);5、10、20 mJ·s/cm2 UVB照射後48 h細胞的生存率分彆為(84.9±1.2)％、(69.3±17.4)％和(32.8±4.5)％,均明顯低于對照組的(100.0±0.0)％,差異均有統計學意義(P=0.047、0.000、0.000);5、10、20 mJ·s/cm2 UVB照射後72 h細胞的生存率分彆為(55.1±3.0)％、(42.1±1.9)％和(26.1±4.7)％,與對照組的(100.0±0.0)％相比生存率明顯降低,差異均有統計學意義(均P=0.000).JC-1染色法檢測錶明,對照組細胞內可見紅色熒光,5mJ·s/cm2 UVB照射後細胞內齣現綠色熒光,10 mJ·s/cm2及20 mJ·s/cm2 UVB照射組齣現綠色熒光增彊,紅色熒光減弱.5、10、20 mJ· s/cm2 UVB照射後細胞內的ROS由0.4％分彆增加至35.8％、51.9％和76.7％.0 mJ·s/cm2 UVB照射組凋亡和壞死細胞比例為2.0％,5、10、20 mJ·s/cm2 UVB照射組凋亡和壞死細胞率分彆為4.2％、7.6％和15.1％.10 mJ·s/cm2和20 mJ·s/cm2 UVB照射組細胞內Ca2+水平分彆為0 mJ·s/cm2照射組的(1.2±0.1)倍和(1.3±0.1)倍,差異均有統計學意義(P=0.039、0.004).5、10和20 mJ·s/cm2 UVB照射組HLE B-3細胞PMCA3 mRNA錶達量均明顯低于對照組,差異均有統計學意義(P=0.001、0.004、0.000),各組細胞中的PMCA蛋白相對錶達量也均明顯低于對照組,差異均有統計學意義(P=0.000、0.000、0.001). 結論 UVB照射可導緻白內障髮生的機製可能與人LECs中PMCA3錶達量下降和誘導LECs凋亡有關,UVB的作用呈劑量和時間依賴性.
배경 세포질막개ATP매3(PMCA3)삼여유지정상체상피세포(LECs) Ca2+적평형,가능여백내장적병리과정유관,자외선B(UVB)시인기백내장적중요인소지일,단UVB대LECs중PMCA3표체적영향소유보도. 목적 연구UVB대인LECs계B-3(HLE B-3) PMCA3표체적영향. 방법 대HLE B-3세포진행배양화전대,당세포체80％이상융합시분별폭로우용불동제량(0、5、10、20 mJ·s/cm2)적UVB분별조사0、20、40화80 s,연후계속배양24、48화72 h,용MTT법검측불동제량UVB조사후세포적생존솔;용JC-1염색법검측UVB조사후세포선립체막전위(△ψm)적변화;이DCFH-DA염색법검측UVB조사후세포내활성양족(ROS)적변화;채용annexin V-FITC/PI염색법검측UVB조사후세포적조망정황;용Fluo-3/AM염색법검측세포내Ca2+농도적변화;분별채용실시형광정량PCR(real-time PCR)법화Western blot법검측UVB조사후세포중PMCA3 mRNA급PMCA단백적표체변화. 결과 수착UVB조사제량적증가화조사시간적연장,세포생존솔균명현하강,차이균유통계학의의(F분조=72.411,P=0.000; F시간=36.588,P=0.000),기중10 mJ·s/cm2、20 mJ·s/cm2 UVB조사후24 h HLE B-3세포적생존솔분별위(75.3±2.2)％화(48.7±4.5)％,명현저우대조조적(100.0±0.0)％,차이균유통계학의의(P=0.001、0.000);5、10、20 mJ·s/cm2 UVB조사후48 h세포적생존솔분별위(84.9±1.2)％、(69.3±17.4)％화(32.8±4.5)％,균명현저우대조조적(100.0±0.0)％,차이균유통계학의의(P=0.047、0.000、0.000);5、10、20 mJ·s/cm2 UVB조사후72 h세포적생존솔분별위(55.1±3.0)％、(42.1±1.9)％화(26.1±4.7)％,여대조조적(100.0±0.0)％상비생존솔명현강저,차이균유통계학의의(균P=0.000).JC-1염색법검측표명,대조조세포내가견홍색형광,5mJ·s/cm2 UVB조사후세포내출현록색형광,10 mJ·s/cm2급20 mJ·s/cm2 UVB조사조출현록색형광증강,홍색형광감약.5、10、20 mJ· s/cm2 UVB조사후세포내적ROS유0.4％분별증가지35.8％、51.9％화76.7％.0 mJ·s/cm2 UVB조사조조망화배사세포비례위2.0％,5、10、20 mJ·s/cm2 UVB조사조조망화배사세포솔분별위4.2％、7.6％화15.1％.10 mJ·s/cm2화20 mJ·s/cm2 UVB조사조세포내Ca2+수평분별위0 mJ·s/cm2조사조적(1.2±0.1)배화(1.3±0.1)배,차이균유통계학의의(P=0.039、0.004).5、10화20 mJ·s/cm2 UVB조사조HLE B-3세포PMCA3 mRNA표체량균명현저우대조조,차이균유통계학의의(P=0.001、0.004、0.000),각조세포중적PMCA단백상대표체량야균명현저우대조조,차이균유통계학의의(P=0.000、0.000、0.001). 결론 UVB조사가도치백내장발생적궤제가능여인LECs중PMCA3표체량하강화유도LECs조망유관,UVB적작용정제량화시간의뢰성.
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.
