Characterization of Immortalized Human Corneal Endothelial Cell Line Cultivated on Human Amniotic Membrane (HAM) for Toxicological Test

Abstract

Abstract: : Purpose: To establish human corneal endothelial cell line (IHCEn) by transducing HPV E6 and E7 oncogenes and to evaluate inherent toxicological properties of functional genes cultivate on HAM. Methods:Primary human corneal endothelial cells (PHCEn) were infected with a recombinant retrovirus harboring HPV E6 and E7, and the transformed cells were clonally selected by G418. These cells were cultured on a flask and lyophilized human amnion membrane (LAM). Messenger RNA expression pattern of channel proteins, essential for maintaining corneal hydration, were screening by RT–PCR (VDAC2, VDAC3, CLCN3, SLC4A4, Na⁺/K⁺ ATPase. etc.) and immunohistochemical staining (Na⁺/K⁺ ATPase, etc.). Results:Successful immortalization was confirmed by stable expressions of HPV E6 and E7 oncogenes. The various genes related human endothelial cell were expressed in both a flask and LAM. Immunohistochemical staining pattern of Na⁺/K⁺ ATPase exhibited a more strongly expressed on cultivated LAM than a flask. Futhermore, the cell density of LAM culture more increased than a flask culture. Lyophilized AM serves as a good substrate for corneal endothelial cell line. Conclusions: These results suggest that human corneal endothelial cell line on HAM could be used to examine the endothelial function for toxicological test. Acknowledgment: This work was supported by a grant of the Korean Health 21 R&D Project, Ministry of Health & Welfare, Republic of Korea. (01–PJ1–PG4–01PT02–0002)

Keywords: cornea: endothelium