Lipid-mediated gene transfer of acidic fibroblast growth factor into human corneal endothelial cells.
The aim of this study was to optimize non-viral gene transfer conditions and investigate the effect of fibroblast growth factor-1 (FGF-1) gene transfer on human corneal endothelial cell (HCEC) proliferation. Five non-viral vectors (Lipofectin, DMRIE-C, DAC-30, Effectene, FuGene6) were used to transfect HCEC with plasmids coding for enhanced green fluorescent protein (EGFP) and FGF-1. Transfection efficiency and toxicity (n=6) were quantified and optimized using the EGFP construct by FACS-analysis. Using optimal conditions HCEC were transfected with the FGF-1 plasmid and cell proliferation as well as expression of FGF-1 were determined at days 4 and 7 by counting and western blotting, respectively. Lipofectin (17+/-2.02%) transfected HCEC more successfully than DMRIE-C (11+/-1.46%), Effectene (9+/-0.62%), FuGene (9+/-0.93%) and DAC-30 (7+/-0.59%). Toxicity of the lipids ranged from 2 to 4%. Optimal HCEC proliferation was achieved with DAC-30/FGF-1 (P<0.05), whereas all other vectors did not result in significantly increased cell proliferation. However, all of the transfected cells produced FGF-1 in different amounts as indicated by western blotting. Efficient and almost non-toxic transfer of the FGF-1 gene into HCEC can be successfully achieved by lipid-based techniques. Using optimal conditions significantly increased cell proliferation was independent on gene transfer efficiency. This may indicate that even a low transfection rate is sufficient to produce a concentration of FGF-1 that will have a stimulatory effect on HCECs.
Dannowski
Haike
H
Department of Ophthalmology, Charité-Universitätsmedizin Berlin, Campus Virchowklinikum, Augustenburger Platz 1, Berlin D-13353, Germany.
Bednarz
Jürgen
J
Reszka
Regina
R
Engelmann
Katrin
K
Pleyer
Uwe
U
D015153:Blotting, Western / Q000379:methods; D002452:Cell Count; D002455:Cell Division / Q000235:genetics; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000235:genetics; D042783:Endothelial Cells / Q000502:physiology / Q000648:ultrastructure; D004728:Endothelium, Corneal / Q000502:physiology* / Q000648:ultrastructure; D016220:Fibroblast Growth Factor 1 / Q000032:analysis / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008055:Lipids / Q000235:genetics; D008081:Liposomes; D008854:Microscopy, Electron / Q000379:methods; D010714:Phosphatidylethanolamines / Q000235:genetics; D000644:Quaternary Ammonium Compounds; D014162:Transfection / Q000379:methods*
C498175:Effectene; C411955:FuGene; D008055:Lipids; D008081:Liposomes; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D016220:Fibroblast Growth Factor 1; C070648:3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol; C087276:(3-dimyristyloxypropyl)(dimethyl)(hydroxyethyl)ammonium; C020888:1,2-dielaidoylphosphatidylethanolamine; D002784:Cholesterol