摘要
目的检测过氧化氢诱导的人晶状体上皮细胞HLE-B3生物活性及长链非编码RNA-p21(lncRNA-p21)的表达变化。方法将HLE-B3细胞分为正常对照组和过氧化氢组,分别用正常培养液和含200μmol/L过氧化氢的培养液培养24 h。采用MTS比色法检测细胞活力;采用活性氧试剂盒检测细胞活性氧簇(ROS)的水平;采用流式细胞术检测细胞凋亡情况;采用Caspase-3活性试剂盒法检测细胞Caspase-3活性;采用Western Blot方法检测细胞凋亡相关Bax,Bcl-2蛋白表达;采用流式细胞术检测细胞周期分布;采用EDU增殖试剂盒检测细胞增殖能力;采用实时荧光定量PCR法检测细胞中lncRNA-p21的表达;采用荧光原位杂交实验法检测细胞中lncRNA-p21的定位。结果过氧化氢组细胞ROS水平为4.65±0.38,明显高于正常对照组的1.00±0.01,差异具有统计学意义(t=16.66,P<0.05)。与正常对照组相比,过氧化氢组细胞凋亡率显著上升,Caspase-3活性增强,凋亡蛋白Bax相对表达量明显升高,差异均有统计学意义(t=20.69、39.80、12.73,均P<0.05)。与正常对照组相比,过氧化氢组G2期细胞比例明显增多,差异有统计学意义(t=23.10,P<0.05)。过氧化氢组EDU阳性细胞比例为(25.41±6.99)%,明显低于正常对照组的(50.58±9.15)%,差异具有统计学意义(t=6.559,P<0.05)。过氧化氢组lncRNA-p21相对表达量为2.36±0.29,明显高于正常对照组的1.02±0.02,差异具有统计学意义(t=7.893,P<0.05)。荧光原位杂交实验结果显示,lncRNA-p21定位于细胞质中。结论过氧化氢诱导的氧化应激模型中晶状体上皮细胞增殖能力显著降低、凋亡水平显著上升,ROS和lncRNA-p21表达水平升高。lncRNA-p21可能参与晶状体上皮细胞的氧化应激损伤过程。
Objective To detect the changes in the biological activity and expression of long-chain non-coding RNA-p21(lncRNA-p21)in human lens epithelial cells HLE-B3 damage induced by hydrogen peroxide.Methods HLE-B3 cells were divided into normal control group and hydrogen peroxide group,which were cultured in normal culture medium and culture medium containing 200μmol/L hydrogen peroxide for 24 hours,respectively.Cell viability was determined by MTS colorimetric method.Cellular reactive oxygen species(ROS)level was detected using ROS assay kits.Cell apoptosis was tested by flow cytometry.Cell Caspase-3 activity was detected using Caspase-3 assay kit.Expressions of Bax and Bcl-2 proteins related to cell apoptosis were determined by Western Blot.Cell cycle distribution was determined by flow cytometry.Cell proliferation ability was detected by EDU proliferation assay kit.The expression of lncRNA-p21 in cells was detected by real time fluorescence quantitative polymerase chain reaction(PCR).The localization of lncRNA-p21 in cells was detected by fluorescence in situ hybridization.Results The ROS content of cells in hydrogen peroxide group was(4.65±0.38),significantly higher than(1.00±0.01)of normal control group,and the difference was statistically significant(t=16.66,P<0.05).Compared with the normal control group,the cell apoptosis rate was significantly increased,the activity of Caspase-3 was enhanced,and the relative expression of Bax was significantly increased in the hydrogen peroxide group,with statistically significant differences(t=20.69,39.80,12.73,all at P<0.05).Compared with the normal control group,the proportion of G2 phase cells in the hydrogen peroxide group significantly increased,showing a statistically significant difference(t=23.10,P<0.05).The EDU-positive cell rate of hydrogen peroxide group was(25.41±6.99)%,significantly lower than(50.58±9.15)%of normal control group(t=6.559,P<0.05).The relative expression level of lncRNA-p21 in the hydrogen peroxide group was 2.36±0.29,significantly higher than 1.02±0.02 in the normal control group(t=7.893,P<0.05).The fluorescence in situ hybridization experiments indicate that lncRNA-p21 was localized in the cytoplasm.Conclusions In the oxidative stress model induced by hydrogen peroxide,the proliferation ability of lens epithelial cells significantly decreases,the apoptosis level significantly increases,and the expression levels of ROS and lncRNA-p21 enhances.lncRNA-p21 may be involved in the oxidative stress injury process of lens epithelial cells.
作者
董晓鸣
刘雨轩
纪力旸
王静
张劲松
Dong Xiaoming;Liu Yuxuan;Ji Liyang;Wang Jing;Zhang Jinsong(Shenyang Aier Excellent Ophthalmology Hospital,Aier Group Ophthalmology Hospital Group Cataract and Artificial Lens Research Institute,Shenyang Aier Ophthalmology Precision Medical Research Institute,Ophthalmology Department of the Fourth Affiliated Hospital of China Medical University,China Medical University Ophthalmology Hospital,Liaoning Provincial Key Laboratory of Lens Research,Shenyang 110000,China;Aier College of Ophthalmology,Central South University,Changsha 410000,China)
出处
《中华实验眼科杂志》
CAS
CSCD
北大核心
2024年第3期232-239,共8页
Chinese Journal Of Experimental Ophthalmology
基金
国家自然科学基金(8187040881)
湖南省自然科学基金(2021JJ40003)
沈阳市科学技术计划公共卫生研发专项(21-173-9-12)
沈阳市中青年科技创新人才支持计划项目(RC210388)。
