目的:观察661W细胞低氧条件下基因表达水平和信号通路的早期改变，寻找潜在功能性靶基因。方法:将培养的小鼠661W细胞分为低氧处理组、常氧对照组。低氧处理组细胞置于37 ℃、体积分数分别为1%、5% COn 2的三气培养箱中培养；常氧对照组细胞置于37 ℃、体积分数为5% COn 2培养箱中培养。培养4 h后，采用高通量测序技术对两组661W细胞进行转录组测序，筛选显著差异表达基因（DEG ）。对筛选得到的DEG进行聚类热图分析、基因注释（GO）功能富集分析、京都基因与基因组百科全书（KEGG）的信号通路富集分析和蛋白互作网络（PPI）分析。采用逆转录聚合酶链反应（RT-PCR）对DEG mRNA表达进行验证。n 结果:共筛选出506个DEG，其中上调基因459个，下调基因47个。GO功能富集分析结果显示，DEG主要富集的生物过程是细胞对低氧反应、糖酵解、细胞增生负调控及凋亡等。KEGG信号通路富集分析结果显示，糖酵解与糖异生、果糖代谢、磷酸戊糖途径以及淀粉和蔗糖代谢等糖代谢途径被显著富集。缺氧诱导因子（HIF）-1α信号通路、糖酵解、FoxO信号通路、胰岛素信号通路和腺苷酸活化蛋白激酶（AMPK）信号通路参与了上述过程。PPI分析结果显示，低氧相关的关键基因是n Aldoa、n Aldoc、n Gpi1、n Hk2、n Hk1、n Pfkl、n Pfkp、n Vhl、n Fbxo10、n Fbxo27。RT-PCR检测结果显示，低氧处理组细胞中15个DEG mRNA表达水平均较常氧对照组升高，差异有统计学意义（n P<0.05）。N-myc下游调控基因-1（n Ndrg1）、n Mt1及血管内皮生长因子A（n VEGFA） mRNA表达水平有低氧时间依赖性。n 结论:低氧下DEG主要为糖代谢相关基因、细胞对缺氧反应相关基因以及调控增生和凋亡相关基因。HIF-1α信号通路、糖酵解、FoxO信号通路和AMPK信号通路参与了低氧下661W细胞的早期改变。n Aldoa、n Aldoc、n Gpi1、n Hk2、n Hk1、n Pfkl、n Pfkp、n Vhl、n Fbxo10、n Fbxo27可能在661W细胞应对低氧反应中起到关键作用。n Ndrg1、n Mt1及n VEGFA可作为研究缺血、缺氧相关眼底疾病的潜在功能性靶基因。n “,”Objective:To analyze the early changes of gene expression levels and signaling pathways in 661W cell line under hypoxic conditions and to find potential functional target genes.Methods:The cultured mouse 661W cells were divided into hypoxia treatment group and normoxia control group. Cells in the hypoxia treatment group were cultured in a three-gas incubator with volume fraction of 1% and 5% COn 2 at 37 ℃. Cells in the normoxia control group were cultured in an incubator at 37 ℃ with volume fraction of 5% COn 2. High-throughput sequencing technology was used to sequence the transcriptome of 661W cell treated with hypoxia and normoxia for 4 hours to screen for differentially expressed genes (DEG). Clustering heat map analysis, gene ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network (PPI) analysis were performed. The reverse transcription-polymerase chain reaction (RT-PCR) was used to verify the accuracy of the sequencing results.n Results:A total of 506 differentially expressed genes were screened, including 459 up-regulated genes and 47 down-regulated genes. GO functional enrichment analysis showed that the main biological processes of DEG were the cell's response to hypoxia, glycolysis, negative regulation of cell proliferation and apoptosis. hypoxia inducible factor (HIF)-1α pathway, glycolysis, Forkhead box O (FoxO) pathway, Insulin signaling pathway and Adenosine 5′-monophosphate-activated protein kinase (AMPK) pathway were involved in the above process. PPI analysis results showed that hub genes related to hypoxia weren Aldoa, n Aldoc, n Gpi1, n Hk2, n Hk1, n Pfkl, n Pfkp, n Vhl, n Fbxo10 and n Fbxo27. The RT-PCR results showed that the relative expression levels of 15 DEG mRNA in the hypoxic treatment group were higher than that of the normoxic control group, and the difference was statistically significant (n P<0.05). The mRNA expression levels of N-myc downstream-regulated gene-1 (n Ndrg1 ), n Mt1, and vascular endothelial growth factor A (n VEGFA) were time-dependent on hypoxia.n Conclusions:Under hypoxia, DEG is mainly related to glucose metabolism, cell response to hypoxia, regulation of proliferation and apoptosis. HIF-1α pathway, glycolysis, FoxO pathway and AMPK pathway are involved in the early changes of 661W cells under hypoxia.n Aldoa, n Aldoc, n Gpi1, n Hk2, n Hk1, n Pfkl, n Pfkp, n Vhl, n Fbxo10, n Fbxo27 may play key roles in the response of 661W cells to hypoxia. n Ndrg1, n Mt1 and n VEGFA could be potential functional target genes for the study of ischemia and hypoxia-related fundus diseases.n