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
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国家自然科学基金(No.81370997)


Discrepant gene expression profile and phenotype changes in human lens epithelial cells after oxidative damage
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National Natural Science Foundation of China(No. 81370997)

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    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H2O2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-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,H2O2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。

    Abstract:

    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-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,TP53I3,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 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.

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梅林,王嵩,闫博,等.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.国际眼科杂志, 2016,16(5):822-825.

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  • 收稿日期:2016-02-22
  • 最后修改日期:2016-04-11
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  • 在线发布日期: 2016-05-03
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