摘要
目的探讨生理低氧条件下与常规氧环境下培养的人晶状体上皮细胞(human lens epithelial cell,HLEC)在抗氧化能力及基因表达谱上的差异。方法 HLEC传代培养24 h后,分别置于体积分数21%常氧培养箱、体积分数1%低氧培养箱孵育24 h,消化、超速离心后收集细胞上清分别检测SOD、CAT活性以及GSH含量,Trizol提取细胞总RNA进氉行基因表达谱检测,生物信息学软件DAVID分析差异基因的功能聚类,qRT-PCR验证差异基因。结果低氧组细胞的SOD、CAT酶活性及GSH含量均低于常氧组,差异均有统计学意义(均为P<0.05)。表达谱芯片结果显示:符合差异表达标准的基因中,在低氧组细胞上调的基因有319个,下调的基因有83个。Gene Ontology分析显示:319个上调基因聚类于148个功能分类中,富集值最高的功能分类为低氧反应。选取5个上调基因进行qRT-PCR验证显示:DDIT4、VEGFA、EDN1、VLDLR、EGLN1在低氧组中的表达量均高于常氧组,差异均有统计学意义(均为P<0.05)。结论生理低氧状态下HLEC的抗氧化能力弱于常氧组,两种氧环境下细胞的基因表达谱不同,提示相应的抗氧化损伤机制可能存在差异。
Objective To compare the anti-oxidative capability and gene expres- sion profile of human lens epithelial cells （HLEC） cultured in both physiological hypox- ia and normoxia. Methods After 24hour subculture, HLEC were cultured in hypoxia and atmospheric oxygen respectively for another 24 hours. After trypsinized and centri- fuged, HLEC supematant was assayed for the amount of reduced glutathione （GSH） and activities of superoxide dismutase （SOD） and catalase （CAT）. Total RNA was ex- tracted by Trizol to detect gene expression profile. Bioinformatical database DAVID was used to perform gene ontology （GO） analysis, The differentially expressed genes were validated by qRT-PCR. Results The amount of GSH and activities of SOD and CAT in the hypoxia group were lower than those in the normoxia group （ all P 〈 0.05 ）. Gene ex- pression profile indicated 319 up-regulated genes and 83 down-regulated genes in the hypoxia group. All the up-regulated genes were further enriched in 148 biological processes. Response to hypoxia was the most significant process, among which 5 up- regulated genes were further identified for their expression by qRT-PCR. It was shown that the expression level of DDIT4, VEGFA, EDNl, VLDLR and EGLN1 in the hypoxia group were higher than those in the normoxia group （ all P 〈 0.05 ）. Conclusion HLEC have weaker anti-oxidative ability in physiological hypoxia. Genes are differential- ly expressed in different oxygen environments, which may contribute to differential anti- oxidative mechanisms.
出处
《眼科新进展》
CAS
北大核心
2015年第5期409-412,共4页
Recent Advances in Ophthalmology
基金
国家自然科学基金资助(编号:81370997)~~
关键词
人晶状体上皮细胞
生理低氧
基因表达谱
human lens epithelial cells
physiological hypoxia
gene expressionprofile
