ESTABLISHMENT OF HUMAN LENS EPITHELIAL CELL LINES

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The aim of cataract research is to prevent or delay human cataract formation. Cataract is the world's leading cause of blindness. Impaired epithelial function invariably leads to a loss of transparency. A therapy aimed at delaying or preventing human cataracts needs to be evaluated on human lens epithelial cells (HLECs). Due to the scarcity of viable human lenses, numerous investigators have tried to culture HLECs. However, normal, HLECs complete 2-4 passages in culture and undergo senescence. HLECs have properties different from those of the lower species. For example, lens epithelial lines can be established from lower species using standard techniques; this is not true of HLECs. Metabolic fluxes in various species are also different relative to the polyol pathway. Aldose reductase activity is high in the dog and rat lens, very low in the human lens and absent in the mouse. Although viral transformation of HLECs permits growth, virally transformed cells have properties that are different from their non-transformed counterparts. Experiments are needed to determine the properties of normal HLECs. Our aims are to establish a reliable supply of normal HLECs, characterize them and make them available to the eye research community. This is absolutely necessary for future studies. The aims are to: 1. Determine if a wound-healing environment permits the long-term growth of normal HLECs from young, old and cataractous lenses. 2. Determine if the introduction of telomerase into HLECs permits establishment of lines fromlens explants, permits established lines to overcome senescence, and if the presence of telomerase maintains the diploid state and allows maintenance of in vivo characteristics. Determine the ploidy, isoenzyme phenotyping, DNA fingerprint, alphaA and alphaB crystallin expression, and growth in cells generated Aim 1 or 2. 3. Determine if there are metabolic differences between cells from young, old and cataractous lenses. 4. Identify lens-specific regulatory elements in the promoters of genes that encode gamma-glutamylcysteine synthetase (GCS).