Integrated transcriptomic and proteomic analyses of retinas in mice with laser-induced CNV were performed using RNA sequencing and tandem mass tag. In addition, the laser-treated mice received systemic interferon-β (IFN-β) therapy. Measurements of CNV lesions were acquired by the confocal analysis of stained choroidal flat mounts. The proportions of T helper 17 (Th17) cells were determined by flow cytometric analysis.
A total of differentially expressed 186 genes (120 up-regulated and 66 down-regulated) and 104 proteins (73 up-regulated and 31 down-regulated) were identified. The gene ontology and KEGG pathway analyses indicated that CNV was mainly associated with immune and inflammatory responses, such as cellular response to IFN-β and Th17 cell differentiation. Moreover, the key nodes of the protein-protein interaction network mainly involved up-regulated proteins, including alpha A crystallin and fibroblast growth factor 2, and were verified by Western blotting. To confirm the changes in gene expression, real-time quantitative PCR was performed. Furthermore, levels of IFN-β in both the retina and plasma, as measured by enzyme-linked immunosorbent assay (ELISA), were significantly lower in the CNV group than in the control group. IFN-β treatment significantly reduced CNV lesion size and promoted the proliferation of Th17 cells in laser-treated mice.
This study demonstrates that the occurrence of CNV might be associated with the dysfunction of immune and inflammatory processes and that IFN-β could serve as a potential therapeutic target.
使用RNA测序和串联质量标签对具有激光诱导的CNV的小鼠中的视网膜进行综合转录组和蛋白质组分析。此外,激光治疗的小鼠接受全身干扰素-β(IFN-β)治疗.通过对染色的脉络膜扁平支架的共聚焦分析获得CNV病变的测量。通过流式细胞术分析确定T辅助细胞17(Th17)细胞的比例。
■鉴定了总共186个差异表达基因(120个上调和66个下调)和104个蛋白质(73个上调和31个下调)。基因本体论和KEGG通路分析表明,CNV主要与免疫和炎症反应有关。例如对IFN-β和Th17细胞分化的细胞应答。此外,蛋白质相互作用网络的关键节点主要涉及上调蛋白,包括αA晶状体蛋白和成纤维细胞生长因子2,并通过Western印迹进行验证。为了确认基因表达的变化,进行实时定量PCR。此外,视网膜和血浆中IFN-β的水平,通过酶联免疫吸附试验(ELISA)测量,CNV组明显低于对照组。在激光治疗的小鼠中,IFN-β治疗显着减少了CNV病变的大小并促进了Th17细胞的增殖。
■这项研究表明,CNV的发生可能与免疫和炎症过程的功能障碍有关,IFN-β可以作为潜在的治疗靶点。