Hypoxia Regulated Gene Transcription in Human Optic Nerve Lamina Cribrosa Cells in Culture
Hypoxia Regulated Gene Transcription in Human Optic Nerve Lamina Cribrosa Cells in Culture
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DOI：
10.1167/iovs.11-6729
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Leonard, Martin O.
中科院分区：
文献类型：
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作者：
Kirwan, Ruaidhri P.;Felice, Luca;Leonard, Martin O.
目的。血管灌注不足、细胞外基质重塑和轴突丢失是青光眼性视神经乳头的病理特征。我们报告了一项新的研究，展示了暴露于体外缺氧应激的视神经筛板（LC）细胞的转录反应。人胶质原纤维酸性蛋白 (GFAP) 阴性 LC 细胞的原代培养物由四个供体产生。细胞暴露于缺氧应激 (1% O2) 或常氧 (21% O2) 下 24 小时。使用 Affymetrix HG-U133A 微阵列 (n = 3) 鉴定缺氧反应基因，并通过实时 PCR 进行验证 (n = 3)。通过 ELISA (n = 4) 测量分泌蛋白，通过蛋白质印迹 (n = 4) 测量细胞蛋白。表达数据用NIH DAVID软件注释，并使用Core_TF软件鉴定缺氧反应基因启动子中推定的转录因子位点。结果。缺氧敏感基因包括参与细胞凋亡（例如 BNIP3）、神经发生（例如 STC1）、细胞外基质（例如 MIF、DDR1/TrkE 和 IGFR2）、线粒体（例如 CYP1B1）和血管生成（例如 VEGF）的基因。选定基因的实时 PCR 支持微阵列识别的表达变化。 ELISA 和蛋白质印迹验证了蛋白质生产的相应变化。启动子序列询问揭示了缺氧反应基因启动子中推定的保守转录因子结合位点（例如，HIF 和 CREB）。结论。我们的数据表明，LC 细胞基因表达对体外氧水平降低敏感，并提供了这种反应的潜在转录调节因子的生物信息证据。 （投资眼科可见科学。2012 年；53：2243-2255）DOI：10.1167/iovs.11-6729
PURPOSE. Vascular hypoperfusion, extracellular matrix remodeling and axon loss are pathological characteristics of the glaucomatous optic nerve head. We report a novel study demonstrating transcriptional responses in optic nerve lamina cribrosa (LC) cells exposed to in vitro hypoxic stress.METHODS. Primary cultures of human glial fibrillary acid protein (GFAP) negative LC cells were generated from four donors. Cells were exposed to 24 hours of hypoxic stress (1% O2) or normoxia (21% O2). Hypoxia responsive genes were identified using Affymetrix HG-U133A microarrays (n = 3) and validated with real time PCR (n = 3). Secreted protein was measured by ELISA (n = 4) and cellular protein by Western blot (n = 4). Expression data were annotated with NIH DAVID software and putative transcription factor sites in hypoxia-responsive gene promoters were identified using Core_TF software.RESULTS. Hypoxia-sensitive genes included those involved in apoptosis (e.g., BNIP3), neurogenesis (e.g., STC1), extracellular matrix (e.g., MIF, DDR1/TrkE, and IGFR2), mitochondrion (e.g., CYP1B1) and angiogenesis (e.g., VEGF). Real time PCR for selected genes supported the expression changes identified by microarray. ELISA and Western blot validated corresponding changes in protein production. Promoter sequence interrogation revealed putative conserved transcription factor binding sites (e.g., HIF and CREB) in the promoters of the hypoxia responsive genes.CONCLUSIONS. Our data show that LC cell gene expression is sensitive to reduced oxygen levels in vitro and provides bioinformatic evidence of the potential transcriptional regulators of this response. (Invest Ophthalmol Vis Sci. 2012; 53: 2243-2255) DOI:10.1167/iovs.11-6729