摘要
目的探讨胸腺肽β4(Tβ4)对H_2O_2诱导的兔角膜基质细胞氧化应激损伤及继发的细胞凋亡的抑制作用。方法采用组织块培养法获得原代兔角膜基质细胞,选取传代的兔角膜基质细胞,根据实验目的将细胞分为3组:正常组、H_2O_2组以及治疗组。H_2O_2组细胞用200μmol·L^(-1)的H_2O_2处理4 h,随后更换为不含FBS的DMEM/F-12培养液继续培养细胞48 h。治疗组细胞则是在H_2O_2处理后换用含终浓度为1μg·mL^(-1)Tβ4的无FBS的DMEM/F-12培养液继续培养48 h。同时把按常规方法培养的兔角膜基质细胞设立为正常组。采用CCK-8法检测各组细胞活性;应用2’,7’-二氯荧光黄双乙酸盐探针检测细胞活性氧水平;实时荧光定量PCR检测细胞中过氧化氢酶和锰超氧化物歧化酶mRNA的相对表达量;采用TUNEL法检测细胞凋亡率。ELISA法检测细胞中Caspase-3蛋白的表达。结果组织块培养法培养的兔角膜基质细胞呈多角形,规则排列呈束状。正常组细胞活性为(100.00±0.00)%、H_2O_2组(66.41±9.28)%、治疗组(82.54±7.72)%;H_2O_2组细胞活性明显低于正常组,差异有统计学意义(P<0.01);治疗组细胞活性明显高于H_2O_2组,差异有统计学意义(P<0.01)。正常组细胞活性氧水平为(4.12±0.93)%、H_2O_2组(77.84±6.98)%、治疗组(59.48±8.92)%;与正常组相比,H_2O_2组细胞呈现明显的强阳性着染;H_2O_2组细胞的活性氧水平明显高于正常组,差异有统计学意义(P<0.01);治疗组细胞的活性氧水平明显低于H_2O_2组,差异有统计学意义(P<0.01)。H_2O_2组细胞过氧化氢酶及锰超氧化物歧化酶mRNA的相对表达量较正常组均明显下降,差异均有统计学意义(均为P<0.05);治疗组细胞过氧化氢酶及锰超氧化物歧化酶mRNA的相对表达量较H_2O_2组均显著升高,差异均有统计学意义(均为P<0.05)。正常组细胞凋亡率为(6.81±1.48)%、H_2O_2组(76.14±6.12)%、治疗组(39.04±7.47)%;H_2O_2组细胞凋亡率明显高于正�
Objective To investigate the inhibitory effects of thymosin β4 (Tβ4) on hydrogen peroxide (H2O2)-induced oxidative damage and subsequent cell apoptosis of rabbit corneal keratocytes. Methods Primary rabbit corneal keratocytes were cultured by explant culture technique.After passage,according to the aims of the present study,the rabbit corneal keratocytes were divided into 3 groups:control group,H2O2 group and treatment group.The cells of the H2O2 group were treated with 200 μmol·L^-1 H2O2 for 4 h,then the cells were cultured with FBS-free DMEM/F-12 medium for 48 h.The cells of the treatment group were cultured with FBS-free DMEM/F-12 medium containing 1 μg·mL^-1 Tβ4 for 48 h after treatment with H2O2.Beyond that,control group were designed in which the cells were cultured with routine methods.The cell activity was detected by CCK-8.The 2’,7’-dichlorodihydrofluorescein diacetate was applied to detect the cellular reactive oxygen species (ROS).Detection of the relative level of catalase mRNA and MnSOD mRNA was performed by real-time fluorescent quantitative PCR.The cell apoptosis rate was detected by TUNEL methods.The content of Caspase-3 protein of cells was examined by ELISA assay. Results The primary rabbit corneal keratocytes cultured by explant culture technique were dendritic,and arranged regularly in bundles.The cell activity of control group,H2O2 group,and treatment group were (100.00±0.00)%,(66.41±9.28)%,and (82.54±7.72)%,respectively.The cell activity of the H2O2 group was markedly reduced in comparison with the control group,and the difference was statistically significant ( P <0.01).The cell activity of the treatment group was apparently increased compared to the H2O2 group,and the difference was statistically significant ( P <0.01).The levels of ROS of control group,H2O2 group,and treatment group were (4.12±0.93)%,(77.84±6.98)%,and ( 59.48± 8.92)%,respectively.The cells of the H2O2 group were significantly positive staining compared to the control group.The level of ROS of the H2O2 gro
作者
刘菁华
郝朋
李轩
LIU Jing-Hua;HAO Peng;LI Xuan(Clinical College of Ophthalmology,Tianjin Medical University,Tianjin 300020,China;Tianjin Eye Hospital,Tianjin Eye Institute,Tianjin Key Laboratory of Ophthalmology and Visual Science ,Tianjin 300020,China)
出处
《眼科新进展》
CAS
北大核心
2019年第3期207-212,共6页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:81170828
81670837)
天津市应用基础与前沿技术研究计划项目(编号:15JCZDJC35300)
天津市卫计委科技攻关项目(编号:14KG133)~~
关键词
胸腺肽β4
氧化应激
过氧化氢
细胞凋亡
兔角膜基质细胞
thymosin β4
oxidative stress
hydrogen peroxide
cell apoptosis
rabbit corneal keratocytes
