Human Retinal Pigment Epithelium Cells as Functional Models for the RPE In Vivo
Human Retinal Pigment Epithelium Cells as Functional Models for the RPE In Vivo
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DOI：
10.1167/iovs.11-8021
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发表时间：
2011-11-01
影响因子：
4.4
通讯作者：
Crosson, Craig E.
中科院分区：
文献类型：
--
作者：
Ablonczy, Zsolt;Dahrouj, Mohammad;Crosson, Craig E.
目的.视网膜色素上皮（RPE）的两种最常用的体外模型是胎儿人RPE（fhRPE）和ARPE-19细胞;然而，对其屏障特性的研究产生了相互矛盾的结果。为了比较它们作为RPE模型的实用性，分析了它们的形态和功能特征。两种细胞类型的单层在可渗透膜滤器上生长。屏障功能和细胞形态通过跨上皮阻力（TER）测量和免疫组织化学进行了评估。通过免疫印迹和ELISA测定评估蛋白质表达，并通过HPLC. MTT表征类维生素A代谢。两种培养物均形成紧密连接。然而，只有fhRPE细胞是色素沉着的，大小和形状均匀，表达高水平的RPE标记物，代谢全反式视黄醇，并产生高TER（>400 Omega cm 2）。色素上皮衍生因子（PEDF）的净分泌在两种培养物中均指向顶端，但fhRPE细胞的分泌速率比ARPE-19细胞高1000倍。fhRPE培养物中血管内皮生长因子（VEGF）的净分泌显著较高，并且该分泌的方向是基底外侧;而ARPE-19细胞中的净分泌是顶端。在新鲜培养基中，VEGF-E降低了两种培养物中的TER;然而，在条件培养基中，fhRPE细胞对VEGF-E给药没有反应，但是用抗PEDF抗体再处理条件培养基允许fhRPE细胞对VEGF-E完全反应。fhRPE细胞的特性与体内功能正常的RPE一致，而ARPE-19细胞类似于病理或老化的RPE。这些结果表明，这两种细胞类型在理解RPE功能的不同，特定方面的效用。（Invest Ophthalmol维斯科学。2011;52：8614-8620）DOI：10.1167/iovs.11-8021
PURPOSE. The two most commonly used in vitro models of the retinal pigment epithelium (RPE) are fetal human RPE (fhRPE) and ARPE-19 cells; however, studies of their barrier properties have produced contradictory results. To compare their utility as RPE models, their morphologic and functional characteristics were analyzed.METHODS. Monolayers of both cell types were grown on permeable membrane filters. Barrier function and cellular morphology were assessed by transepithelial resistance (TER) measurements and immunohistochemistry. Protein expression was evaluated by immunoblotting and ELISA assays, and retinoid metabolism characterized by HPLC.RESULTS. Both cultures developed tight junctions. However, only the fhRPE cells were pigmented, uniform in size and shape, expressed high levels of RPE markers, metabolized all-trans retinal, and developed high TER (>400 Omega cm(2)). The net secretion of pigment-epithelium-derived factor (PEDF) was directed apically in both cultures, but fhRPE cells exhibited secretion rates a thousand-fold greater than in ARPE-19 cells. The net secretion of vascular endothelial growth factor (VEGF) was significantly higher in fhRPE cultures and the direction of this secretion was basolateral; while net secretion was apical in ARPE-19 cells. In fresh media, VEGF-E reduced TER in both cultures; however, in conditioned media fhRPE cells did not respond to VEGF-E administration, but retreatment of the conditioned media with anti-PEDF antibodies allowed fhRPE cells to fully respond to VEGF-E.CONCLUSIONS. Properties of fhRPE cells align with a functionally normal RPE in vivo, while ARPE-19 cells resemble a pathologic or aged RPE. These results suggest a utility for both cell types in understanding distinct, particular aspects of RPE function. (Invest Ophthalmol Vis Sci. 2011;52:8614-8620) DOI: 10.1167/iovs.11-8021