CAJ | 학술논문

背景对蓝光照射所致视网膜色素上皮(RPE)细胞损伤与细胞内钙离子含量变化关系的深入研究可能对探讨视网膜变性类疾病的发生机制及防治具有重要意义. 目的建立人RPE细胞蓝光损伤模型,探讨RPE细胞损伤与细胞内钙离子含量变化的关系.方法将人RPE细胞系培养传代,锥虫蓝染色评估细胞活力.第4代人RPE细胞分别用(2000±500) lx蓝光照射3、6、9、12h,终止细胞培养24 h后,采用TUNEL法检测不同时间点细胞的凋亡情况,确定最适宜的光照度.然后将RPE细胞分为无光照组、光照组、硝苯地平组、光照+硝苯地平组、(-)BayK8644组、光照+(-)BayK8644组.除无光照组外,其他各组细胞均用蓝光照射6h,终止细胞培养24h,采用激光扫描共焦显微镜检测细胞内游离钙离子含量,激发光波长为488 nm,发射光波长为505 nm,每组随机选取30个细胞,扫描细胞图像,采用分析软件分析各组细胞内游离钙离子的荧光强度.结果锥虫蓝染色证实RPE细胞的活力均在90％以上,TUNEL染色和免疫组织化学染色证实光照3h组未见细胞凋亡,而光照后6、9、12h发现不同数量的凋亡细胞.硝苯地平组RPE细胞内游离钙离子荧光强度比无光照组和光照+硝苯地平组低,差异均有统计学意义(均P=0.000)；光照组、(-)BayK8644组、光照+(-)BayK8644组RPE细胞内钙离子荧光强度高于无光照组,差异均有统计学意义(均P=0.000)；光照+硝苯地平组与无光照组相比较,(-)BayK8644组与光照+(-)BayK8644组比较,差异均无统计学意义(P=0.339、0.410). 结论光照度(2000±500)1x、光照6h、终止培养24 h为建立蓝光照射致人RPE细胞损伤模型的最适合条件.蓝光照射所致的人RPE细胞损伤与细胞内游离钙离子含量增加有关.
배경 대람광조사소치시망막색소상피(RPE)세포손상여세포내개리자함량변화관계적심입연구가능대탐토시망막변성류질병적발생궤제급방치구유중요의의. 목적 건립인RPE세포람광손상모형,탐토RPE세포손상여세포내개리자함량변화적관계.방법 장인RPE세포계배양전대,추충람염색평고세포활력.제4대인RPE세포분별용(2000±500) lx람광조사3、6、9、12h,종지세포배양24 h후,채용TUNEL법검측불동시간점세포적조망정황,학정최괄의적광조도.연후장RPE세포분위무광조조、광조조、초분지평조、광조+초분지평조、(-)BayK8644조、광조+(-)BayK8644조.제무광조조외,기타각조세포균용람광조사6h,종지세포배양24h,채용격광소묘공초현미경검측세포내유리개리자함량,격발광파장위488 nm,발사광파장위505 nm,매조수궤선취30개세포,소묘세포도상,채용분석연건분석각조세포내유리개리자적형광강도.결과 추충람염색증실RPE세포적활력균재90％이상,TUNEL염색화면역조직화학염색증실광조3h조미견세포조망,이광조후6、9、12h발현불동수량적조망세포.초분지평조RPE세포내유리개리자형광강도비무광조조화광조+초분지평조저,차이균유통계학의의(균P=0.000)；광조조、(-)BayK8644조、광조+(-)BayK8644조RPE세포내개리자형광강도고우무광조조,차이균유통계학의의(균P=0.000)；광조+초분지평조여무광조조상비교,(-)BayK8644조여광조+(-)BayK8644조비교,차이균무통계학의의(P=0.339、0.410). 결론 광조도(2000±500)1x、광조6h、종지배양24 h위건립람광조사치인RPE세포손상모형적최괄합조건.람광조사소치적인RPE세포손상여세포내유리개리자함량증가유관.
Background Investigating the association of blue light-induced damage of retinal pigmenepithelial (RPE) cellwith intracellulaCa2+ conteniimportanfounderstanding the mechanism of retinal disorders.Objective Thistudy wato establish blue light-induced damage model of human RPE celland explore the relationship between the damage of RPE cell and intracellulaCa2+ content.MethodHuman RPE cellwere cultured and passaged.Cell vitality waassayed by trypan blue staining.Fourth-generation cellwere used in these experiments.The cellwere exposed to blue lighwith an intensity of (2000±500)lx fo3,6,9 o12 hours,and the rate of apoptosiwaassayed by TUNEL to assesthe optimal irradiation time focellcultured.The cellwere then randomized into the withouirradiation group,irradiation only group,nifedipine group,ligh+ nifedipine group,(-) BayK8644 group and ligh+ (-) BayK8644 group.The laseconfocal microscope waused to determine the fluorescence intensity of intracellulafree Ca2+ aan excitation wavelength of 488 nm and an emission wavelength of 505 nm.The cell imagewere analyzed using computesoftware.The differenceof fluorescence intensity among the differengroupwere compared by one-way analysiof variance.ResultTrypan blue staining showed thathe viability of RPE cellwamore than 90％ afteculturing and passaging.No apoptoticell waseen aftelighexposure fo3 hours.However,differennumberof apoptoticellappeared aftelighexposure fo6,9 and 12 hours.The fluorescence intensity of intracellulafree Ca2+ in the nifedipine group wasignificantly lowethan thaof the withouirradiation group othe ligh+ nifedipine group(both aP=0.000).Lasescanning confocal microscopy showed thathe fluorescence intensitieof intracellulafree Ca2+ in the irradiation only group,(-) BayK8644 group,ligh+ (-)BayK8644 group were highethan thaof the withouirradiation group,with statistical significancebetween them(all P=0.000).No significandifferencewere found in the fluorescence intensity of intracellulafree Ca2+ between the ligh+ nifedipine group and withouirradiation group awell abetween the (-)BayK8644 group and the ligh+(-) BayK8644 group(P =0.339,P =0.410).ConclusionThe optical conditionfoblue light-induced RPE cell damage were exposure of blue-ligha(2000± 500) lx fo6 hourand culturing the cellfo24 hours.Blue lighexposure can induce damage of human RPE cellprobably by triggering the increase of intracellulafree Ca2+ concentration.