May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Development and characterization of rabbit corneal endothelial cell line
Author Affiliations & Notes
  • J.–S. Shin
    Ophthalmology, Chung–Ang University, Seoul, Republic of Korea
    Biochemistry, Graduate School of Life Sciences and Biotechnology, Seoul, Republic of Korea
  • C.–W. Kim
    Biochemistry, Graduate School of Life Sciences and Biotechnology, Seoul, Republic of Korea
  • J.–C. Kim
    Ophthalmology, Chung–Ang University, Seoul, Republic of Korea
  • Footnotes
    Commercial Relationships  J. Shin, None; C. Kim, None; J. Kim, None.
  • Footnotes
    Support  Korean Ministry of Health and Welfare PJ1–PG4–01PT02–0002
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 4785. doi:
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      J.–S. Shin, C.–W. Kim, J.–C. Kim; Development and characterization of rabbit corneal endothelial cell line . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4785.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To develop rabbit corneal endothelial cell line by transducing human papilloma virus (HPV) type 16 E6 and E7 oncogenes, and to characterize the gene expression profile and Na+–K+ ATPase activity of an established cell line. Methods: Primary rabbit corneal endothelial cells were infected with recombinant retrovirus harboring HPV E6 and E7, and the transformed cells were clonally selected by G418. Corneal endothelial origin of the selected clones was confirmed by RT–PCR and Western blot. Results: Among the total eight independent clones, one cell line (clone no. A3) cultured over 40 passages was chosen to further characterize the inherent biological properties; Typical cell doubling time for these cells was 51 hrs, and mean cell density cultured on flask was 1140 per mm2. Important genes for corneal endothelial functions (collagen type IV, voltage–dependent anion channel (VDAC) 2, VDAC 3, sodium bicarbonate cotransporter (NBC) 1, chloride channel protein (CLCN) 3, potassium channel (KCNC) 3, and KCND 2) were shown to be expressed in comparable level to the normal counterpart. Also, biochemical assay for Na+–K+ ATPase revealed that this cell line has comparable activity to primary cultured normal endothelium. Upon cultured on denuded amniotic membrane, these cells exhibited typical cobble–stone morphology, and mean cell density was significantly increased to 1400 per mm2 compared to that obtained on culture flask. Moreover, these cells lined as a monolayer on amniotic membrane increased the transparency of the amniotic membrane. Conclusions: These results suggest that rabbit corneal endothelial cell lines obtained here maintain normal corneal endothelial characteristics, and amniotic membrane could be a good substrate for corneal endothelium.

Keywords: cornea: endothelium • cornea: basic science • NaK ATPase 
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