Transcriptomic comparison of young and aged choroid at the bulk and single-cell levels (bulk)

Purpose: The goals of this study were to identify cell specific expression patterns from infant and aged choroid, with a particular emphasis on how endothelial gene expression changes with age. Methods: Independent libraries were prepared for infant and adult choroidal cells from seventeen human donors. Trephine punch biopsies were used to acquire central RPE and choroidal tissue (infants: 4 mm punch biopsy; adults: 8 mm hemisected punch biopsy). Poly-A selected RNA transcripts underwent 150 base-pair paired-end sequencing with an Illumina HiSeq 2000. Reads were mapped to the human genome hg19 with STAR (version = 020201) and quantified with the featureCounts function from the Rsubread package (version = 1.28.1). Differential expression analysis was performed with edgeR (version = 3.26.8). Results: Differential expression analysis identified genes enriched in infant versus adult libraries. Differentially expressed genes (p-value < 0.00001, FDR < 0.001, cpm >2, logFC > 1) were input into WebGestalt for over-representation analysis. Aged choroidal samples were enriched in proinflammatory genes. Conclusions: This study provides a transcriptomic comparison of infant and aged human donor choroid at the bulk and single-cell levels. The aged choroid, in particular the aged vasculature, is enriched in pro-inflammatory genes. Overall design: mRNA profiles from infant and adult choroidal isolates were generated from seventeen donor eyes followed by sequencing on a HiSeq 2000.

研究目的：本研究旨在识别婴儿与老年人脉络膜的细胞特异性表达模式，重点关注内皮细胞基因表达随年龄的变化情况。
研究方法：从17名人类供体中分别制备婴儿及成人脉络膜细胞的独立文库。采用环钻活检获取中央视网膜色素上皮（Retinal Pigment Epithelium, RPE）及脉络膜组织：婴儿组使用4mm环钻活检，成人组使用8mm对半切开式环钻活检。经聚腺苷酸（Poly-A）富集的RNA转录本采用Illumina HiSeq 2000平台进行150bp双端测序。使用STAR（版本号020201）将测序读段比对至人类基因组hg19，并通过Rsubread软件包的featureCounts函数（版本1.28.1）完成定量分析。采用edgeR（版本3.26.8）开展差异表达分析。
研究结果：差异表达分析筛选出婴儿文库相较于成人文库富集的基因。将满足p值<0.00001、错误发现率（False Discovery Rate, FDR）<0.001、每百万reads计数（counts per million, cpm）>2、对数倍数变化（log2 Fold Change, logFC）>1的差异表达基因输入WebGestalt进行过度表达富集分析。结果显示，老年脉络膜样本中促炎基因显著富集。
研究结论：本研究在批量与单细胞水平上，对婴儿与老年人类供体脉络膜开展了转录组学比较。老年脉络膜，尤其是老年脉络膜血管系统，存在促炎基因的富集现象。
实验整体设计：从17例供体眼球中分离得到婴儿与成人脉络膜样本，构建mRNA表达谱，随后在Illumina HiSeq 2000平台上完成测序。