Cai Hui, Del Priore Lucian V
Department of Ophthalmology, Harkness Eye Institute, Columbia University, New York, NY, USA.
Mol Vis. 2006 Jan 5;12:1-14.
ARPE19 is a spontaneously immortalized cell line of human retinal pigment epithelium (RPE) that is used widely to draw inferences about the behavior of adult human RPE (ahRPE). We used DNA microarray analysis to compare the gene expression profiles of these two cell types.
Second-passage cultured ahRPE from four human donors (age range 48-82 years) and ARPE19 cultured to confluence in five dishes were used for this DNA microarray study. Total RNA was isolated and first- and second-strand complimentary DNA was synthesized using standard techniques. Biotin-labeled antisense complimentary RNA was produced by an in vitro transcription reaction. Target hybridization, washing, staining, and scanning probe arrays were done following an Affymetrix GeneChip Expression Analysis Manual. Microarray data were normalized and statistical techniques were used to determine the presence or absence of expression of individual genes within ARPE19 and ahRPE, and their relative expression levels.
Hierarchic clustering analysis demonstrated that the gene expression profile of ahRPE and ARPE19 samples cluster into two distinct groups with no discernable overlap. The expression of 5,634+/-65 gene probes (out of 12,600 on microarray Human U95Av2 chip) was detected in ARPE19 cells compared to 5,580+/-84 genes in ahRPE cells from four human donor eyes. Thirty-five genes are expressed exclusively in ahRPE and nine genes exclusively in ARPE19 cells. Fifty additional genes have a threefold increase and 40 genes have a threefold decrease in expression level in ahRPE compared to ARPE19. There was no clear difference in the global expression level of genes known to be related to phagocytosis, angiogenesis, or apoptosis.
There are significant differences in the gene expression profile of ahRPE compared to ARPE19, and with some genes exclusively being expressed in one group and other genes being upregulated or downregulated by threefold. Caution should be exercised when generalizing results obtained from ARPE19 to the behavior of ahRPE.
ARPE19是一种人视网膜色素上皮（RPE）的自发永生化细胞系，广泛用于推断成人RPE（ahRPE）的行为。我们使用DNA微阵列分析来比较这两种细胞类型的基因表达谱。
本DNA微阵列研究使用了来自四名人类供体（年龄范围48 - 82岁）的第二代培养ahRPE以及在五个培养皿中培养至汇合的ARPE19。使用标准技术分离总RNA并合成第一链和第二链互补DNA。通过体外转录反应产生生物素标记的反义互补RNA。按照Affymetrix GeneChip表达分析手册进行靶标杂交、洗涤、染色和扫描探针阵列。对微阵列数据进行归一化处理，并使用统计技术确定ARPE19和ahRPE中各个基因的表达情况及其相对表达水平。
层次聚类分析表明，ahRPE和ARPE19样本的基因表达谱聚为两个不同的组，没有明显重叠。在ARPE19细胞中检测到5,634±65个基因探针（在微阵列Human U95Av2芯片上的12,600个探针中）表达，而来自四名人类供体眼睛的ahRPE细胞中有5,580±84个基因表达。35个基因仅在ahRPE中表达，9个基因仅在ARPE19细胞中表达。与ARPE19相比，另外50个基因在ahRPE中的表达水平增加了三倍，40个基因的表达水平降低了三倍。已知与吞噬作用、血管生成或细胞凋亡相关的基因的整体表达水平没有明显差异。
与ARPE19相比，ahRPE的基因表达谱存在显著差异，一些基因仅在一组中表达，而其他基因上调或下调了三倍。在将从ARPE19获得的结果推广到ahRPE的行为时应谨慎。
