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Xingjun Fan, Jeremy Whitson, Vincent M Monnier, Ulrich Hopfer; Establishment of a lens epithelial monolayer culture system for the study of glutathione transport. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1214.
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In aging, human lens glutathione (GSH) levels are impaired and accompanied with increased oxidation, protein disulfide formation and protein cross-link. Lens GSH concentrations are believed tightly regulated by two main mechanisms, i.e. by adjusting the rate of synthesis and transporter systems. This was confirmed by our LEGSKO mouse (PLOS ONE 2012(7):e50832). However, the molecular identity of GSH transporters has remained elusive; In order to test the candidate GSH transporters, we are establishing a lens epithelial monolayer culture system to mimic in vivo conditions, since lens has a unique structure with a polarized undifferentiated epithelial cell layer and differentiated fibers.
The GSH (H3-labled) uptake and transport was tested in homozygous LEGSKO and wild type whole lenses using ex vivo culture and in human lens epithelial cell line (HLE-B3) and primary cultured mouse lens epithelial cells monolayer that were cultured in 24-well Transwell plates. After two weeks of growth, monolayer integrity was monitored by transepithelial electrical resistance (TEER) and the rejection value of Lucifer yellow, a nontransportable fluorescence compound. The monolayer tight junction was characterized by immunocytochemistry using tight junction marker ZO-1.
GSH uptake in LEGSKO blocked GSH biosynthesis was more than 5x increased supporting the existence of GSH uptake system in lens. For the monolayer culture system, the TEER showed significant elevation starting at day 5 and reached relative stable value (~60ohm) at day 9. The 1-hour LY rejection value reached to 95% at day 12 from 35% at day 5. The tight junction marker ZO-1 was highly expressed at day 12 based on immunocytochemistry stain using ZO-1 antibody. GSH transport assay by applying H3-GSH to apical chamber showed 5% of GSH penetrated through HLE-B3 monolayer at 30min, and reached to 15% at 1 hour. The results indicated that monolayer has dynamic regulation in GSH transporting through the epithelial cells.
The successful establishment of HLE-B3 cells into monolayers is expected the greatly facilitate the identification and characterization of specific GSH transporters into the lens.
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