Krugluger Walter, Seidel Stefan, Steindl Kerstin, Binder Susanne
Department of Clinical Chemistry, The Ludwig Boltzmann Institut of Retinology and Biomicroscopic Lasersurgery, Rudolfstiftung Hospital, Vienna, Austria.
Graefes Arch Clin Exp Ophthalmol. 2007 Oct;245(10):1543-8. doi: 10.1007/s00417-007-0635-0. Epub 2007 Aug 10.
Culture of retinal pigment epithelium (RPE) cells might be a future option in the therapy of various degenerative retinal diseases. However, the molecular changes which occur during in vitro expansion of RPE cells during culture are not fully elucidated. The aim of this study was to evaluate molecular changes in the RPE cell line ARPE-19 after stimulation with different growth factors.
Cultured ARPE-19 cells were stimulated for 72 hours with rh-EGF, rh-IGF-1, rh-VEGF or rh-bFGF, and transcriptional changes of the differentiation markers cytokeratin 18 and RPE65 and of the key molecules of the wnt pathway, beta-catenin, and glycogen synthase kinase-3 (GSK-3) were evaluated by real time RT-PCR.
We found a significant decrease of cytokeratin 18 and RPE65 transcription after stimulation with rh-EGF (0.47 +/- 0.42 and 0.32 +/- 0.57-fold, respectively; p < 0.05). A significant reduction of beta-catenin and GSK-3 mRNA was found in ARPE-19 cells stimulated with rh-IGF-1 (0.61 +/- 0.25 and 0.52 +/- 0.02-fold, respectively) or rh-EGF (0.55 +/- 0.19 and 0.76 +/- 0.26-fold, respectively). No changes of beta-catenin mRNA were observed after stimulation with rh-VEGF or bFGF.
Our data suggest an inhibition of the beta-catenin-pathway in ARPE-19 cells by IGF-1 and EGF, suggesting that ARPE-19 cell proliferation is, at least in part, driven by the beta-catenin pathway. Furthermore, induction of proliferation by EGF results in a loss of differentiation markers in these cells. Maintaining the RPE phenotype is still one of the main problems for RPE- transplantation.
视网膜色素上皮（RPE）细胞培养可能是未来治疗各种视网膜退行性疾病的一种选择。然而，RPE细胞在体外培养扩增过程中发生的分子变化尚未完全阐明。本研究的目的是评估不同生长因子刺激后RPE细胞系ARPE - 19中的分子变化。
用重组人表皮生长因子（rh - EGF）、重组人胰岛素样生长因子 - 1（rh - IGF - 1）、重组人血管内皮生长因子（rh - VEGF）或重组人碱性成纤维细胞生长因子（rh - bFGF）刺激培养的ARPE - 19细胞72小时，通过实时逆转录聚合酶链反应（RT - PCR）评估分化标志物细胞角蛋白18和RPE65以及Wnt信号通路关键分子β - 连环蛋白和糖原合酶激酶 - 3（GSK - 3）的转录变化。
我们发现用rh - EGF刺激后，细胞角蛋白18和RPE65转录显著降低（分别为0.47±0.42倍和0.32±0.57倍；p < 0.05）。在用rh - IGF - 1（分别为0.61±0.25倍和0.52±0.02倍）或rh - EGF（分别为0.55±0.19倍和0.76±0.26倍）刺激的ARPE - 19细胞中，β - 连环蛋白和GSK - 3 mRNA显著减少。用rh - VEGF或bFGF刺激后未观察到β - 连环蛋白mRNA的变化。
我们的数据表明IGF - 1和EGF可抑制ARPE - 19细胞中的β - 连环蛋白信号通路，提示ARPE - 19细胞增殖至少部分由β - 连环蛋白信号通路驱动。此外，EGF诱导的增殖导致这些细胞中分化标志物的丧失。维持RPE表型仍然是RPE移植的主要问题之一。
