Department of Ophthalmology, Ludwig Boltzmann Institute for Retinology and Biomicroscopic Lasersurgery, Rudolf Foundation Clinic, Juchgasse 25, 1030 Vienna, Austria.
Graefes Arch Clin Exp Ophthalmol. 2011 Aug;249(8):1195-200. doi: 10.1007/s00417-011-1673-1. Epub 2011 Apr 15.
To analyze whether epidermal growth factor (EGF) exerts regulatory effects on proliferation and differentiation in ARPE19 cells after different incubation periods (24 vs. 48 h) for obtaining ideal conditions for feasible rejuvenation and autologous transplantation of retinal pigment epithelial cells (RPE cells).
To evaluate gene expression patterns of RPE-specific differentiation and proliferation markers as well as transcriptional and translational changes of beta-catenin (ß-catenin)-signaling markers by fluorescence activated cell sorting (FACS) and reverse transcription - polymerase chain reaction (RT-PCR) after 24 h of EGF treatment.
After 24 h of EGF treatment, a significant decrease of retinal pigment epithelium-specific protein 65 (RPE 65), cellular retinaldehyde-binding protein (CRALBP) and cytokeratin 18 in ARPE-19 cells was scaled. In addition, an increase of cyclin D1 expression and a significant decrease of glycogen synthase kinase-3beta (GSK-3ß) and beta-catenin (ß-catenin) were equally observed after 24 and 48 h of EGF treatment. Cell-cycle studies revealed an increase of ARPE cells in S-G2/M phase after 24 h of EGF treatment.
Our data demonstrate the induction of proliferation and upregulation of the ß-catenin signaling pathway by EGF even after 24 h of incubation. As ideal cell culture conditions are essential for maintaining RPE-specific phenotypes, short incubation times enhance RPE cell quality for feasible rejuvenation and subsequent autologous transplantation of RPE cells.
分析表皮生长因子（EGF）在不同孵育时间（24 小时与 48 小时）后对 ARPE19 细胞增殖和分化的调节作用，以获得可行的视网膜色素上皮（RPE）细胞再生活跃和自体移植的理想条件。
通过荧光激活细胞分选（FACS）和逆转录-聚合酶链反应（RT-PCR），评估 RPE 特异性分化和增殖标志物的基因表达模式，以及转录和翻译β-连环蛋白（β-catenin）信号标志物的变化，在 EGF 处理 24 小时后。
EGF 处理 24 小时后，ARPE-19 细胞中视网膜色素上皮特异性蛋白 65（RPE65）、细胞视网膜结合蛋白（CRALBP）和细胞角蛋白 18 的表达显著减少。此外，在 EGF 处理 24 和 48 小时后，同样观察到 cyclin D1 表达增加，糖原合酶激酶-3β（GSK-3β）和β-catenin（β-catenin）表达显著减少。细胞周期研究表明，EGF 处理 24 小时后 ARPE 细胞进入 S-G2/M 期的比例增加。
我们的数据表明，EGF 甚至在孵育 24 小时后即可诱导增殖和上调β-catenin 信号通路。由于理想的细胞培养条件对于维持 RPE 特异性表型至关重要，因此短的孵育时间可提高 RPE 细胞质量，从而实现可行的 RPE 细胞再生活跃和随后的自体移植。
