Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line
Effects of culture conditions on heterogeneity and the apical junctional complex of the ARPE-19 cell line
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DOI：
10.1167/iovs.06-0166
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发表时间：
2006-08-01
影响因子：
4.4
通讯作者：
Rizzolo, Lawrence J.
中科院分区：
文献类型：
--
作者：
Luo, Yan;Zhuo, Yehong;Rizzolo, Lawrence J.
目的. ARPE-19是被广泛研究的人RPE的自发转化细胞系。本报告探讨其适用于研究的RPE的紧密连接。将ARPE-19维持在标准培养基或三种减少血清的培养基制剂之一中。通过免疫细胞化学、免疫印迹分析和逆转录-聚合酶链反应检测细胞骨架蛋白和连接蛋白的表达和分布。屏障功能测定为跨上皮电阻（TER）和辣根过氧化物酶（HRP）的跨单层扩散。与使用第15代至第20代细胞的原始报告不同，ARPE-19的常用菌株表现出细长和多边形细胞的异质混合物。肌动蛋白分布在应力纤维而不是环向带。TER低，HRP通透性高。claudins和cytokeratins的表达是异质性的。部分分化可以通过操纵生长培养基在细胞亚群中诱导。一个共同的影响是增加JAM-A，AF-6和PAR-3的表达与肌动蛋白丝的重新分布。这种效应伴随着HRP渗透性降低10倍，但对TER的影响极小。ARPE-19的性质似乎正在以可能取决于细胞如何维持和传代的方式发生变化。在比较实验室之间的数据和解释只有一部分细胞可能对实验刺激产生反应的研究时，应谨慎行事。专门媒体促进成熟的粘附连接，但只有部分成熟的紧密连接。
PURPOSE. ARPE-19 is a spontaneously transformed cell line of human RPE that is widely studied. This report examines its suitability for studying the tight junctions of the RPE.METHODS. ARPE-19 was maintained in standard medium or one of three reduced-serum medium formulations. The expression and distribution of cytoskeletal and junctional proteins were examined by immunocytochemistry, immunoblot analysis, and the reverse transcription-polymerase chain reaction. Barrier function was measured as the transepithelial electrical resistance (TER) and the transmonolayer diffusion of horseradish peroxidase (HRP).RESULTS. Unlike the original reports using passage-15 to -20 cells, commonly available strains of ARPE-19 exhibited a heterogeneous mixture of elongate and polygonal cells. Actin was distributed in stress fibers rather than circumferential bands. The TER was low, and the permeability of HRP was high. The expression of claudins and cytokeratins was heterogeneous. Partial differentiation could be induced in subsets of cells by manipulating the growth medium. A common effect was an increase in the expression of JAM-A, AF-6, and PAR-3 that correlated with a redistribution of actin filaments. This effect was accompanied by a 10 x decrease in the permeability of HRP, but a minimal effect on TER.CONCLUSIONS. The properties of ARPE-19 appear to be changing in ways that may depend on how the cells are maintained and passaged. Caution should be exercised in comparing data between laboratories and in interpreting studies in which only a subset of cells may respond to experimental stimuli. Specialized media promoted the maturation of the adherens junction, but only a partial maturation of the tight junctions.