目的：探讨人晶状体上皮细胞( hulan 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H2 O2刺激。24 h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-qPCR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-qPCR结果证实,6个主要参与促凋亡或抗凋亡调节的基因,包括 BCL2 A1、TP53 I3、FAS、ZMAT3、DDB2和BCL2 L1,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,H2 O2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。%AlM: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 H2 O2 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 DAVlD. The elevated expressions of up -regulated genes in HLEC after oxidative stimulation were confirmed by RT-qPCR. The apoptosis of HLEC induced by oxidative damage was detected using 3 - ( 4, 5 -dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide( MTT) assay and flow cytometry. RESULTS: Gene expression profiling assay demonstrated that 367 genes were up-regulated in HLEC after oxidative stimulation. These genes were enriched in 310 biological processes mainly associated with p53 -related signaling pathways, apoptosis, programed cell death and etc. Six genes mainly pro- or anti-apoptotic, including BCL2A1, TP53l3, FAS, ZMAT3, DDB2 and BCL2L1, were confirmed to be up-regulated by oxidative stimulation using RT-qPCR(P<0. 05). Results of MTT assay and flow cytometry showed that apoptosis of HLEC gradually appeared after cells were treated with H2 O2 and became the main consequence of oxidative stimulation. CONCLUSlON:Oxidative stimulation can induce up-regulation of proapoptotic genes and lead to apoptosis of HLEC, even though antiapoptotic genes can also be promoted.
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