Nagymihály Richárd, Veréb Zoltán, Albert Réka, Sidney Laura, Dua Harminder, Hopkinson Andrew, Petrovski Goran
Stem Cells and Eye Research Laboratory, Department of Ophthalmology, Faculty of Medicine, University of Szeged, Szeged, Hungary.
Academic Department of Ophthalmology, Division of Clinical Neuroscience, University of Nottingham, Nottingham, UK.
Clin Exp Ophthalmol. 2017 Jul;45(5):509-519. doi: 10.1111/ceo.12903. Epub 2017 Feb 14.
The study aims to characterise human corneal endothelial cell (HCEnC) cultures generated by the peel-and-digest method based on their surface protein/carbohydrate expression pattern.
Quantitative polymerase chain reaction was used to compare expression of vimentin, CD90, Cytokeratin-19, ZO-1 and Claudin 14 in cultured HCEnC and cell line B4G12 versus stromal cells. Fluorescence-activated cell sorting was used to assess surface protein distribution of cultured and uncultured HCEnC. Distribution of surface proteins/carbohydrates was visualised by immunofluorescent and lectin staining.
Human corneal endothelial cell and B4G12 showed lower expression level for vimentin, CD90, Cytokeratin-19 compared with stromal cells; while ZO-1 was expressed in endothelial cells, Claudin 14 was detected in B4G12 only. Fluorescence-activated cell sorting analyses revealed CD166, CD47, CD44, CD54, CD73, CD90, CD105, CD106, CD112, CD146 and CD325 to be present, with CD34 to be absent from cultured HCEnC. Freshly isolated, non-cultivated HCEnCs were CD90, CD73, CD146 and CD325 positive. Carbohydrates were detected by lectins LCA, PHA E, PHA L, PSA, sWGA, Con A, RCA 120 and WGA, but cultured HCEnC showed negative for GSL I, SBA, DBA, PNA and UEA I.
Cultures established by the peel-and-digest method are probably not prone to stromal contamination, but the cells are likely to undergo endothelial-to mesenchymal transition as suggested by apparent morphological changes.
本研究旨在根据人角膜内皮细胞（HCEnC）通过剥离消化法培养所得细胞的表面蛋白/碳水化合物表达模式对其进行特征描述。
采用定量聚合酶链反应比较培养的HCEnC和细胞系B4G12与基质细胞中波形蛋白、CD90、细胞角蛋白-19、紧密连接蛋白-1（ZO-1）和克劳丁14的表达。利用荧光激活细胞分选技术评估培养和未培养的HCEnC的表面蛋白分布。通过免疫荧光和凝集素染色观察表面蛋白/碳水化合物的分布。
与人角膜内皮细胞和B4G12相比，基质细胞中波形蛋白、CD90、细胞角蛋白-19的表达水平较低；紧密连接蛋白-1在内皮细胞中表达，而克劳丁14仅在B4G12中检测到。荧光激活细胞分选分析显示培养的HCEnC中存在CD166、CD47、CD44、CD54、CD73、CD90、CD105、CD106、CD112、CD146和CD325，而不存在CD34。新鲜分离的未培养HCEnC为CD90、CD73、CD146和CD325阳性。通过凝集素荆豆凝集素（LCA）、木菠萝凝集素E（PHA E）、木菠萝凝集素L（PHA L）、花生凝集素（PSA）、小麦胚凝集素（sWGA）、刀豆球蛋白A（Con A）、蓖麻凝集素120（RCA 120）和小麦胚芽凝集素（WGA）检测到碳水化合物，但培养的HCEnC对神经节苷脂I（GSL I）、大豆凝集素（SBA）、双花扁豆凝集素（DBA）、花生凝集素（PNA）和荆豆凝集素I（UEA I）呈阴性。
通过剥离消化法建立的培养物可能不易受到基质污染，但从明显的形态变化来看，细胞可能会发生内皮-间充质转化。
