ROCK抑制剂Y-27632在体外培养的兔角膜内皮细胞紫外线损伤修复中的作用  

Roles of ROCK inhibitor Y-27632 on rabbit corneal endothelial cells against ultraviolet radiation-induced damage in vitro

作　　者：朱娉[1] 姜志昕[1] 郝朋[1] 赵堪兴[1] 李轩[1] ZHU Ping;JIANG Zhixin;HAO Peng;ZHAO Kanxing;LI Xuan(Tianjin Eye Hospital,Tianjin Key Laboratory of Ophthalmology and Vision Science,Tianjin Eye Institute,Clinical College of Ophthalmology,Tianjin Medical University,Affiliated Eye Hospital of Nankai University,Tianjin 300020,China)

机构地区：[1]天津市眼科医院,天津市眼科学与视觉科学重点实验室,天津市眼科研究所,天津医科大学眼科临床学院,南开大学附属眼科医院,天津市300020

出　　处：《眼科新进展》2021年第3期201-205,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号81170828);天津市应用基础与前沿技术研究计划项目(编号15JCZDJC35300);天津市卫生行业重点攻关项目(编号14KGl33)。

摘　　要：目的探讨ROCK抑制剂Y-27632在兔角膜内皮细胞(rabbit corneal endothelial cells,RCECs)紫外线损伤修复中的作用。方法收集30只健康成年新西兰大白兔的角膜,显微镜下撕除角膜后弹力层和内皮层,用胰蛋白酶消化法获得原代RCECs。选取第2代处于对数生长期的RCECs分为三组A组为正常对照组,RCECs为常规培养液培养;B组为紫外线照射组,RCECs经紫外线照射20 s制备细胞损伤模型,常规内皮细胞培养液培养;C组为紫外线照射+Y-27632组,在细胞损伤模型中加入终浓度10μmol·L^(-1)的Y-27632细胞培养液培养。采用MTT比色法检测各组细胞活性。采用Transwell迁移实验检测各组细胞的迁移能力。利用BrdU细胞增殖实验检测各组细胞的增殖能力。运用细胞黏附实验检测各组细胞黏附性。采用实时荧光定量PCR法检测各组细胞周期蛋白(CyclinD1)和细胞周期蛋白依赖激酶抑制因子(CDKN1B)mRNA的相对表达量。Western blot法检测各组CyclinD1和细胞周期蛋白依赖激酶(Cdk2)的表达情况。结果MTT比色法检测结果显示,B组细胞的活性(80.61±3.40)%低于A组(100%)和C组(92.55±4.56)%,差异均有统计学意义(P=0.000、0.025)。Transwell迁移实验结果显示,B组迁移细胞数目(39.3±4.4)个/高倍视野较A组(77.0±6.7)个/高倍视野和C组(54.3±3.4)个/高倍视野减少,差异均有统计学意义(P=0.023、0.015)。BrdU细胞增殖实验检测结果显示,B组细胞光密度值(0.24±0.05)较A组(0.35±0.08)和C组(0.30±0.07)降低,差异均有统计学意义(P=0.001、0.015)。细胞黏附实验结果显示,B组细胞黏附性(0.183±0.010)较A组(0.541±0.010)和C组(0.353±0.030)下降,差异均有统计学意义(P=0.001、0.000)。实时荧光定量PCR检测结果显示,与A组相比,B组的CyclinD1 mRNA相对表达量(0.49±0.10)下降,CDKN1B mRNA相对表达量(1.25±0.20)上调,差异均有统计学意义(P=0.003、0.002);C组CyclinD1 mRNA的相对表达量(0.80±0.12)比B组(0.49±0.10)增加,CObjective To investigate the roles of ROCK inhibitor Y-27632 on rabbit corneal endothelial cells(RCECs)from ultraviolet(UV)radiation-induced damage in vitro.Methods The corneas of 30 healthy adult New Zealand white rabbits were collected,the Descemet’s membrane was tore off under a microscope,and primary RCECs were obtained by trypsin digestion.The second generation of RCECs in the logarithmic growth phase were collected and divided into three groupsgroup A was the normal control group,and RCECs were cultured in conventional medium,group B was UV radiation-induced RCECs damage group,and RCECs were irradiated with ultraviolet for 20 s,then cultured in conventional endothelial cell culture medium,group C was the ultraviolet irradiation+Y-27632 group,RCECs were irradiated with ultraviolet for 20 s,then cultured in culture medium with Y-27632 at the final concentration of 10μmol·L^(-1).The cell viability of RCECs was examined by MTT assay.Cell migration was measured by Transwell chamber experiment.The proliferation of RCECs was evaluated by BrdU cell proliferation assay.The cell adhesion of RCECs was assessed by cell adhesion assay.The Real-time fluorescence quantitative PCR was used to detect the relative expression levels of CyclinD1 mRNA and CDKN1B mRNA in cells.Western blot was used to detect the protein expression of CyclinD1 and Cdk2 in cells.Results The results of MTT test showed that the cell viability of group B(80.61±3.40)%was lower than that of group A(100%)and group C(92.55±4.56)%,and the differences were statistically significant(P=0.000,0.025).The results of the Transwell migration experiment showed that the number of migrating cells in group B(39.3±4.4)cells/high magnification was lower than that of group A(77.0±6.7)cells/high magnification and group C(54.3±3.4)cells/high magnification,and the differences were statistically significant(P=0.023,0.015).The results of BrdU cell proliferation experiment showed that the cell proliferation ability of group B(0.24±0.05)was lower than that of group A

关 键 词：角膜内皮细胞 ROCK抑制剂 增殖 黏附性 细胞周期蛋白 细胞周期蛋白依赖激酶 细胞周期蛋白依赖激酶抑制因子 

分 类 号：R771[医药卫生—眼科]