ARPE-19, a human retinal pigment epithelial cell line with differentiated properties
ARPE-19, a human retinal pigment epithelial cell line with differentiated properties
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DOI：
10.1006/exer.1996.0020
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发表时间：
1996-02-01
影响因子：
3.4
通讯作者：
Hjelmeland, LM
中科院分区：
文献类型：
--
作者：
Dunn, KC;AotakiKeen, AE;Hjelmeland, LM
视网膜色素上皮(RPE)在脊椎动物视网膜相邻光感受器的发育和维持中起着至关重要的作用。这项研究描述了ARPE-19的发展和特征，ARPE-19是一种自发生长的人类RPE细胞系，具有正常的核型，在多孔过滤器支架上形成极化上皮单分子层。该细胞系是通过选择性胰酶消化原代RPE培养建立的，导致高度上皮细胞的均匀群体，显示出强大的生长潜力。用Northern分析检测RPE特异性标志物CRALBP和RPE65的表达，以确定细胞的生化分化程度。在ARPE-19总mRNA中检测到1.6kb的CRALBP mRNA转录本和2.8kb的RPE65转录本。Western印迹分析检测CRALBP蛋白在ARPE-19细胞裂解产物中的表达，免疫细胞化学方法检测融合培养的大多数细胞(而非全部细胞)胞浆中CRALBP蛋白的表达。实验确定了单层形成的基本标准，发现ARPE-19细胞在血清含量较低的介质中接种于涂有层粘连蛋白的Transwell-Col滤膜上时，表现出形态极化。通过记录单层细胞的跨上皮阻力来确定紧密连接形成的时间进程，培养4周后达到最大值50-100欧米伽厘米(2)。通过测量H-3-菊糖从细胞培养室的顶端到基侧隔室的通量来评价ARPE-19单层的屏障性能。最后，通过连续稀释法获得ARPE-19克隆亚系，以获得具有高跨上皮阻力(TER)的亚系。亚系的形态多样，克隆的细胞在培养中有衰老的趋势，证实该细胞系未转化。没有亚系单层的TER大于亲本细胞记录的TER。结果表明，ARPE-19细胞具有体内RPE细胞的结构和功能特征，为视网膜色素上皮生理学的体外研究提供了有价值的细胞系。(C)1996年学术出版社有限公司
The retinal pigment epithelium (RPE) plays a critical role in the development and maintenance of adjacent photoreceptors in the vertebrate retina. This study describes the development and characterization of ARPE-19, a spontaneously arising human RPE cell line with normal karyology which forms polarized epithelial monolayers on porous filter supports. The cell line was established by selective trypsinization of a primary RPE culture resulting in a uniform population of highly epithelial cells which exhibit a strong growth potential. To determine the extent of biochemical differentiation, the expression of the RPE-specific markers CRALBP and RPE65 was examined by Northern analysis. A single 1.6 kb CRALBP mRNA transcript and a single 2.8 kb RPE65 transcript were detected in samples of ARPE-19 total mRNA. The expression of CRALBP protein in ARPE-19 cell lysate was detected by Western blot analysis and immunocytochemistry was used to detect CRALBP throughout the cytoplasm of most, but not all, cells in confluent cultures. The essential criteria for monolayer formation were determined experimentally and it was found that ARPE-19 cells exhibit morphological polarization when plated on laminin-coated Transwell-COL filters in medium with a low serum content. The time course of tight-junction formation was determined by recording the transepithelial resistance of monolayers and reached a maximum of 50-100 Omega cm(2) after 4 weeks of culture. Barrier properties of ARPE-19 monolayers were evaluated by measuring the flux of H-3-inulin from the apical to the basolateral compartment of cell culture chambers. Finally, ARPE-19 clonal sublines were generated by serial dilution in an attempt to produce a subline with a high transepithelial resistance (TER). The morphology of the sublines was variable and the cloned cells exhibited a tendency to senesce in culture, confirming that this cell line is not transformed. No subline monolayers developed a TER greater than those recorded for the parent cells. Our results demonstrate that ARPE-19 has structural and functional properties characteristic of RPE cells in vivo and suggest that this cell line will be valuable for in vitro studies of retinal pigment epithelium physiology. (C) 1996 Academic Press Limited