高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析

RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells under high glucose condition

目的观察高糖状态下视网膜血管内皮细胞基因表达谱的RNA-Seq分析结果。方法体外培养视网膜血管内皮细胞，取对数生长期细胞用于实验。将细胞分为对照组、高糖组，两组细胞分别采用5、25 mmol/L的葡萄糖持续培养48 h。应用RNA-Seq技术对两组细胞进行全转录组测序，获得生物学大数据，并在此基础上分析差异表达基因；通过GO功能、Pathway显著性富集分析对差异基因的功能及所在的信号通路进行解释说明。结果对照组和高糖组之间共有449个差异表达基因。其中，上调基因297个，下调基因152个。差异基因功能涉及分子生物学过程调控以及细胞的能量代谢、蛋白合成等诸多方面，其中ITGB1BP2、NCF1、UNC5C等与炎症的产生相关；AKR1C4、ATP1A3、CHST5、LCTL等与细胞的能量代谢相关；DAB1、PRSS55等与蛋白合成相关；SMAD9、BMP4等与细胞外基质的代谢相关。GO功能显著性富集分析结果显示，差异基因的功能主要可以划分为生物学行为调控，细胞组分形成以及分子功能三大板块，主要集中表现在生物学过程部分的系统形成和调节多细胞有机体的形成等。Pathway显著性富集分析前20条具有明显差异的通路，发现差异表达比较明显的是转化生长因子-β（TGF-β）信号通路、补体通路等，色氨酸、丝氨酸、氰氨酸等与氨基酸代谢相关的通路也受到了影响。其中，白细胞抑制因子9、骨形成蛋白4通过TGF-β信号通路发挥作用。结论高糖通过破坏视网膜血管内皮细胞的跨膜传导、细胞外基质代谢以及蛋白质的转录和翻译等，从而影响视网膜血管内皮细胞的功能。

ObjectiveTo observe RNA-Seq analysis of gene expression profiling in retinal vascular endothelial cells after anti-vascular endothecial growth factor （VEGF） treatment.MethodsRetinal vascular endothelial cells were cultured in vitro, and the logarithmic growth phase cells were used for experiments. The cells were divided into the control group and high glucose group. The cells of two groups were cultured for 5 hours with 5, 25 mmol/L glucose, respectively. And then, whole transcriptome sequencing approach was applied to the above two groups of cells through RNA-Seq. Now with biological big data obtained as a basis, to analyze the differentially expressed genes （DEGs）. And through enrichment analysis to explain the differential functions of DEGs and their signal pathways.ResultsThe gene expression profiles of the two groups of cells were obtained. Through analysis, 449 DEGs were found, including 297 upregulated and 152 downregulated ones. The functions of DEGs were influenced by regulations over molecular biological process, cellular energy metabolism and protein synthesis, etc. Among these genes, ITGB1BP2, NCF1 and UNC5C were related to production of inflammation; AKR1C4, ATP1A3, CHST5, LCTL were related to energy metabolism of cells; DAB1 and PRSS55 were related to protein synthesis; SMAD9 and BMP4 were related to the metabolism of extracellular matrix. GO enrichment analysis showed that DEGs mainly act in three ways： regulating biological behavior, organizing cellular component and performing molecular function, which were mainly concentrated in the system generation of biological process part and regulation of multicellular organisms. Pathway enrichment analysis showed that gene expressions of the two cell groups were differentiated in transforming growth factor-β （TGF-β） signaling pathway, complement pathway and amino acid metabolism-related pathways have also been affected, such as tryptophan, serine and cyanide. Among them, leukocyte inhibitory factor 9 and bone morphogenetic protein 4 play a role through the TGF-β signaling pathway.ConclusionsHigh glucose affects the function of retinal vascular endothelial cells by destroying transmembrane conduction of retinal vascular endothelial cells, metabolism of extracellular matrix, and transcription and translation of proteins.