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Impressum
The Effect of Lipid-based Gene Transfer of Acidic Fibroblast Growth Factor (aFGF) on Human Corneal Endothelial Cell (HCEC) proliferation in vitro.
1Dannowski H., 2Bednarz J., 3Reszka R., 2Engelmann K., 1Pleyer U.
1Department of Ophthalmology, Charité Campus Virchow, Humboldt University, Berlin/Germany, 2Department of Ophthalmology, Hamburg- Eppendorf, Hamburg/Germany, 3Max Delbrück Centrum for Molecular Medicine, Berlin-Buch/Germany
Objective: The purpose of our study was to examine the effect
of aFGF on HCEC proliferation in vitro after lipid-mediated gene transfer
and stimulation with exogenous aFGF.
Methods: A previous described cell line (Bednarz et al. 2000) was
cultured and transfected with a plasmid coding for aFGF using different
lipid-based transfection reagents (DAC30, Fugene, Lipofectin, DMRIE, Effectene,
Superfect). The optimal lipid:DNA ratio was detected by gene transfer
of the reporter gene ß-gal. HCEC were seeded at 30 000 cells/well
in 24 well plates. At days 4 and 7 after transfection the cells were counted.
As controls we used cells under standard conditions and cells stimulated
with exogenous aFGF in different concentrations.
Results: Marked differences in gene transfer efficiency were present
using the various transfection reagents. A limited transfection was observed
using DAC30, whereas the other vehicles showed 10-30% ß-gal expression.
Interestingly gene transfer of aFGF by all vectors showed a comparable
increase of cell proliferation of the HCEC (approximate 150%). This was
comparable with the effect of 10ng exogenous aFGF in the controls.
Conclusion: Gene transfer is a promising approach to target corneal
endothelial cells. HCEC are able to increase their proliferation by gene
transfer of aFGF but the capacity for stimulation is limited up to 150%.
The highest proliferation rate was independent from the gene transfer
efficiency and comparable with 10ng of exogenous aFGF. That indicates
that even a low transfection rate is sufficient for a stimulating protein
concentration.
Supported by DFG (Pl 150/10-1, Re 93/7-1)