Effect of HNF-1β ectopic expression on morphological changes and gene expression of dedifferentiated hRPTECs.

Human RPTECs were stimulated with 3 ng/ml TGF-β1 for 48 h followed by re-stimulation with fresh TGF-β1 for 72 h. After replacement with fresh TGF-β1, the hRPTECs were infected with 3.0 MOI Ad-HNF1B or Ad-LacZ. Representative fluorescence microscopy photographs of hRPTECs show non-stimulation (A), TGF-β1 stimulation for 48 h (B), TGF-β1 stimulation for 48 h followed by treatment with TGF-β1 and 3.0 MOI of Ad-LacZ for 72 h (C), and TGF-β1 stimulation for 48 h followed by treatment with TGF-β1 and 3.0 MOI of Ad-HNF1B for 72 h (D). Scale bar = 30 μm. Human RPTECs were stimulated with 3 ng/ml TGF-β1 for 48 h, followed by re-stimulation with fresh TGF-β1 for 72 h. After replacement with fresh TGF-β1, the hRPTECs were infected with 0.1 to 3.0 MOI Ad-HNF1B or Ad-LacZ. The levels of mRNA encoding γ-GT1 (E), claudin-2 (F), type I collagen (G), and fibronectin (H) in the differentiated hRPTECs were determined by real-time RT-PCR. Each column shows the data from non-stimulation (white), TGF-β1 stimulation for 48 h (black), TGF-β1 stimulation for 48 h followed by treatment with TGF-β1 and Ad-LacZ for 72 h (hatched line), and TGF-β1 and Ad-HNF1B for 72 h (dot). Each column and bar presents the mean ± SD from three independent experiments. Statistical significance: # P < 0.05, ## P < 0.01, ### P < 0.001 vs. 48-h TGF-β1-treated group (black column); ** P < 0.01, *** P < 0.001 vs. corresponding TGF-β1- and Ad-LacZ-treated groups (hatched column) by t-tests.