Establishment of a Lens Epithelial Cell Line from a Canine Mature Cataract

Abstract

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 aB-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.

Analysis of Cationic Amino Acid Transport Activity in Canine Lens Epithelial Cells

“…The canine lens epithelial cell line derived from a mature cataract was maintained as described previously [18]. The RNA samples for RT-PCR analysis were obtained from a healthy male Shiba dog (six years old).…”

“…Arginine uptake of LEC was measured as described previously [18]. Briefly, the cells were plated in 5 × 10 5 cells/6-well plate 24 h before the experiment.…”

“…In spite of the importance of cationic amino acids on the ocular surface, little information is available regarding cationic amino acid transport activity in canine LECs. Previously, we developed a lens epithelial cell line originated from a mature cataract of a dog and reported several characteristics of this cell line [18]. In the present study, we investigated the cationic amino acid transport activity of the lens epithelial cell line and determined the cDNA sequence of canine CAT1 as the first step in clarifying the relationship between CAT and the cationic amino acid metabolism on the canine ocular surface.…”

Distribution, Molecular Structure and Functional Analysis of Carnitine Transporter (SLC22A5) in Canine Lens Epithelial Cells

“…The canine lens epithelial cell line originating from mature cataract was maintained as described previously [12]. Several tissues were obtained from a 5-year-old male Japanese Shiba dog after deep anesthesia by pentobarbital.…”

“…In spite of the significance of carnitine on the ocular surface, there has been no report on carnitine transport activity in LECs. Previously, we developed the canine lens epithelial cell line and reported several of its characteristics [12]. In this study, we examined the carnitine transport activity of this cell line, determined its molecular structure, and investigated the distribution of canine SLC22A5.…”