Gene expression profile of conjunctival epithelial cell lines

Human conjunctival cell lines are useful tools for modeling ocular surface disease and evaluation of ocular drugs. Here we demonstrate that the IOBA-NHC and the ChWK conjunctival epithelial cell lines show, using an unbiased gene microarray approach, unique gene expression signatures that differ from primary conjunctival epithelial cells (PCEC) and conjunctival tissue. Globally, the expression profile obtained with the Affymetrix U133A chip (>22000 genes) from PCEC was clustered more closely to conjunctival tissue than either of the 2 cell lines. However, when restricted to Gene Ontology sub-categories: cellular defense, viral replication/cycling, antigen presentation, anti-oxidant pathways and ubiquitin ligase complex, the cell lines correlated reasonably well to PCEC (r > 0.70). In the category response to inflammation, correlation of cell lines to PCEC was poor (r = -0.012 and –0.041 for IOBA-NHC and ChWK respectively). In general, the expression profile in IOBA-NHC cells was better correlated to PCEC than the ChWK cells. This was statistically significant (p<0.05) when one considers all the genes on the chip, or for proteins in the extracellular region, response to wounding, stress, lipid, protein and organic acid metabolism, development and differentiation. Our results are useful for the choice of conjunctival cell lines, if necessary, in future experiments, to increase validity of extrapolation to clinical scenarios. Keywords: Cell type comparison Affymetrix U133A Genechip Experimental samples: IOBA-NHC cells (5 samples) Chang conjunctival epithelial cells WK derivative (4 samples) Primary conjunctival epithelial cells from explants (3 samples, obtained from cadaveric human explants) Conjunctival tissue from pterygium study where small piece uninvolved conjunctiva harvested (4 patients' RNA pooled to form one sample, total number of samples: 4) RT and hybridisation 16 hr according to Affymetrix protocol Labeling with biotin Washing microfluidics station 450 Analysis with Genespring GX 7.3.1 RMA normalisation following by normalisation to chip level median signal These processed data used for correlation analysis Further gene level normalisation to primary conjunctival epithelial cells samples for the purpose of fold change analysis to compare expression in IOBA or ChWK cells vs primary conjunctival epithelial cells.

人结膜细胞系是模拟眼表疾病以及评估眼部药物的重要工具。本研究通过无偏倚基因芯片分析方法，证实IOBA-NHC与ChWK结膜上皮细胞系具有独特的基因表达特征，该特征与原代结膜上皮细胞（primary conjunctival epithelial cells，PCEC）及结膜组织存在显著差异。整体而言，通过Affymetrix U133A芯片（覆盖超22000个基因）获取的PCEC表达谱，与结膜组织的聚类相关性较两种细胞系均更高。但当限定于基因本体论（Gene Ontology）亚类时，包括细胞防御、病毒复制/周期、抗原呈递、抗氧化通路及泛素连接酶复合物，两种细胞系与PCEC的相关性均较为良好（相关系数r>0.70）。而在炎症应答这一亚类中，两种细胞系与PCEC的相关性则较差（IOBA-NHC与ChWK的相关系数r分别为-0.012与-0.041）。总体而言，IOBA-NHC细胞的表达谱与PCEC的相关性优于ChWK细胞。当分析芯片上所有基因，或细胞外区域蛋白、损伤应答、应激、脂质/蛋白质及有机酸代谢、发育与分化相关的基因集时，该差异均具有统计学意义（p<0.05）。本研究结果可为未来实验中结膜细胞系的选择提供参考，从而提升向临床场景外推结果的有效性。
关键词：细胞类型比较、Affymetrix U133A基因芯片
实验样本：IOBA-NHC细胞（5份样本）；ChWK细胞（即Chang结膜上皮细胞WK衍生株，4份样本）；外植体来源的原代结膜上皮细胞（3份样本，取自人体尸体外植体）；翼状胬肉研究中获取的未受累结膜组织（收集4名患者的RNA混合为1份样本，总样本量为4份）
实验流程：依照Affymetrix标准方案进行逆转录（Reverse Transcription，RT）与杂交，反应时长16小时；采用生物素标记样本；使用微流体工作站450完成洗涤步骤；通过Genespring GX 7.3.1进行数据分析；先实施RMA标准化，再以芯片水平的中位信号进行二次标准化。经上述处理的数据用于相关性分析；为开展差异倍数分析以对比IOBA-NHC或ChWK细胞与原代结膜上皮细胞的表达水平，进一步以原代结膜上皮细胞样本为参照，对基因水平的数据实施标准化处理。