Abstract
The aim of this study was to establish a lens epithelial cell (LEC) line originated from a cataract of a dog. An anterior capsulorhexis specimen from a dog naturally developing mature cataracts was obtained prior to routine phacoemulsification cataract extraction. The primary lens epithelial cells were transfected with expression plasmid DNA encoding the large T antigen of replication origin-defective simian virus 40 (SV40), and then a colony was cloned using a glass cylinder. The primary cells stopped proliferation in three passages, while the transfected cells remained proliferative. Functional analysis of Na-dependent vitamin C transporter (SVCT) indicated that the Km value toward ascorbic acid (vitamin C) was 19.9 ± 2.8 μM, and RT-PCR analysis showed that SVCT2 was observed in this cell line while SVCT1 was not, which is one of the characteristics of LECs. Western blot analysis and cytoimmunochemistry indicated immortalized cells produced a protein with a molecular mass of 25 kDa, which reacted with an antibody to αB-crystallin within the whole cytosol. The cloned cell line, termed cdLEC, grew well and could be propagated over 250 times by basically splitting at 1:20. These results indicate that cdLEC may also provide a useful in vitro system for the study of the pathophysiology of cataract.
