Proulx Stéphanie, Landreville Solange, Guérin Sylvain L, Salesse Christian
Unité de Recherche en Ophtalmologie, Centre de Recherche du CHUQ, Pavillon CHUL, Faculté de médecine, Université Laval, Salle S-5, 2705 Boul. Laurier, Ste-Foy, Que., Canada G1V 4G2.
Exp Eye Res. 2004 Aug;79(2):157-65. doi: 10.1016/j.exer.2004.03.004.
Primary cultures of human retinal pigment epithelium (RPE) requires young human donors with short post-mortem time and no known retinal diseases. The use of an established human RPE cell line, like ARPE-19, would be a welcomed alternative to primary cultures. This cell line retains many of the characteristics of RPE cells, including cell morphology, functional tight junctions and expression of CRALBP and RPE65. This study was conducted in order to investigate integrin alpha5 expression at both the gene and protein level in the ARPE-19 cell line and compare the results with those obtained with primary cultures of RPE cells. The potential use of this cell line as a substitute for primary cultures of RPE cells was also considered. Integrin alpha5 protein was detected on RPE and ARPE-19 cultures at different confluencies by immunofluorescence and immunoprecipitation analyses. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to study alpha5 mRNA levels. Transient transfections were performed in order to compare alpha5 promoter strength in both types of cells. Immunofluorescence studies showed that both primary RPE and ARPE-19 cells yielded similar alpha5 staining patterns at all cell confluencies. Both immunoprecipitation and RT-PCR analyses provided evidence that sub-confluent and confluent RPE and ARPE-19 cells have similar cell surface alpha5 protein and mRNA levels whereas post-confluent cells had a marked decrease in both protein and transcript levels. ARPE-19 cells show a large increase in promoter strength compared to primary cultures. When compared to primary cultures, the cell line exhibited major differences in the way the alpha5 promoter is regulated, even if both cell types are cultured under identical conditions. This study demonstrates that primary cultures of human RPE and ARPE-19 cells show reductions in both the alpha5 protein and the mRNA when cells reach post-confluency. However, major differences have been observed in the strength of the alpha5 promoter between both cell types. We also show that culturing ARPE-19 cells in a different growth medium alters the transcriptional activity directed by the alpha5 promoter.
人视网膜色素上皮（RPE）原代培养需要年轻的人类供体，且死后时间短且无已知视网膜疾病。使用已建立的人RPE细胞系，如ARPE - 19，将是原代培养的一个受欢迎的替代方法。该细胞系保留了许多RPE细胞的特征，包括细胞形态、功能性紧密连接以及CRALBP和RPE65的表达。进行这项研究是为了调查ARPE - 19细胞系中整合素α5在基因和蛋白质水平的表达，并将结果与RPE细胞原代培养获得的结果进行比较。还考虑了该细胞系作为RPE细胞原代培养替代品的潜在用途。通过免疫荧光和免疫沉淀分析在不同汇合度的RPE和ARPE - 19培养物中检测到整合素α5蛋白。使用半定量逆转录 - 聚合酶链反应（RT - PCR）研究α5 mRNA水平。进行瞬时转染以比较两种细胞类型中α5启动子的强度。免疫荧光研究表明，原代RPE细胞和ARPE - 19细胞在所有细胞汇合度下产生相似的α5染色模式。免疫沉淀和RT - PCR分析均提供证据表明，亚汇合和汇合的RPE细胞及ARPE - 19细胞具有相似的细胞表面α5蛋白和mRNA水平，而后汇合细胞的蛋白质和转录本水平均显著下降。与原代培养相比，ARPE - 19细胞的启动子强度大幅增加。与原代培养相比，即使两种细胞类型在相同条件下培养，该细胞系在α5启动子的调控方式上也表现出主要差异。这项研究表明，当细胞达到汇合后，人RPE原代培养物和ARPE - 19细胞中的α5蛋白和mRNA均会减少。然而，在两种细胞类型之间观察到α5启动子强度存在主要差异。我们还表明，在不同生长培养基中培养ARPE - 19细胞会改变α5启动子指导的转录活性。
