氧化损伤诱导的人晶状体上皮细胞系基因表达谱的差异和表型改变  

Discrepant gene expression profile and phenotype changes in human lens epithelial cells after oxidative damage

作　　者：梅林[1] 王嵩[1] 闫博[2] 杨安钢[2] 赵晶[2] 严宏[1] 

机构地区：[1]第四军医大学唐都医院眼科,中国陕西省西安市710038 [2]第四军医大学基础部生物化学与分子生物学教研室,中国陕西省西安市710032

出　　处：《国际眼科杂志》2016年第5期822-825,共4页International Eye Science

基　　金：国家自然科学基金(No.81370997)~~

摘　　要：目的:探讨人晶状体上皮细胞(human lens epithelial cells,HLEC)氧化刺激后基因表达谱的差异以及相应的表型改变。方法:培养人晶状体上皮细胞系HLE-B3并给予H_2O_2刺激。24h后,提取细胞总RNA进行基因表达谱芯片检测,并采用生物信息学数据库DAVID对氧化刺激组相比对照组显著上调的基因进行Gene Ontology(GO)功能富集分析。RT-q PCR对上调的基因进行验证。通过MTT和流式细胞仪检测细胞凋亡水平。结果:表达谱芯片结果显示,氧化刺激造成HLEC中367个基因上调,GO分析表明这些基因富集于310个功能类别中,主要包括p53信号通路、细胞凋亡通路、细胞程序性死亡通路等。RT-q PCR结果证实,6个主要参与促凋亡或抗凋亡调节的基因,包括BCL2A1、TP53I3、FAS、ZMAT3、DDB2和BCL2L1,在氧化刺激后表达水平明显上升。MTT实验和流式细胞仪检测结果显示,H_2O_2刺激后HLEC凋亡逐渐上升,是细胞氧化损伤的主要表现。结论:氧化刺激可同时诱导HLEC中促凋亡基因和抗凋亡基因表达水平上调,但最终仍然导致了细胞凋亡。AIM ： To explore the discrepant gene expression profiles and the related phenotype changes in human lens epithelial cells（ HLEC） after oxidative stimulation.METHODS： Human lens epithelial cell line（ HLE- B3）were cultured in normal condition or with H2O2 for 24h.Total RNA were extracted for gene expression profiling assay and gene ontology analysis was performed for the significantly up- regulated genes using bioinformational database DAVID. The elevated expressions of up-regulated genes in HLEC after oxidative stimulation were confirmed by RT- q PCR. The apoptosis of HLEC induced by oxidative damage was detected using 3-（ 4,5-dimethyl- 2- thiazolyl）- 2,5- diphenyl- 2- H-tetrazoliumbromide（ M TT） assay and flowcytometry. RESULTS： Gene expression profiling assay demonstrated that 367 genes were up- regulated in HLEC after oxidative stimulation. These genes were enriched in310 biological processes mainly associated with p53-related signaling pathways, apoptosis, programed cell death and etc. Six genes mainly pro- or anti- apoptotic,including BCL2A1,TP53I3,FAS,ZM AT3,DDB2 and BCL2L1,were confirmed to be up- regulated by oxidative stimulation using RT- q PCR（ P〈0. 05）. Results of M TT assay and flowcytometry showed that apoptosis of HLEC gradually appeared after cells were treated with H2O2 and became the main consequence of oxidative stimulation.CONCLUSION： Oxidative stimulation can induce up-regulation of proapoptotic genes and lead to apoptosis of HLEC, even though antiapoptotic genes can also be promoted.

关 键 词：人晶状体上皮细胞 氧化损伤 表达谱芯片 细胞凋亡 

分 类 号：R776.1[医药卫生—眼科]