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Y. Ryu, J. Ahn, H. Kim, J. Park, J. Kim; Characterization of Immortalized Human Corneal Fibroblasts and Epithelial Cells Cultivated on Human Amniotic Membrane With Collagen Scaffold . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4979.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish human corneal fibroblasts and epithelial cell line by transducing HPV E6 and E7 oncogenes, and to characterize the inherent biological properties of the established cell lines cultivated on human amniotic membrane (AM) with collagen scaffold for developing toxicological test. Methods: Primary human corneal fibroblasts (PHCF) and epithelial cells (PHCEp) were infected with recombinant retrovirus harboring HPV E6 and E7, and clonally selected by G418. Infected genes, HPV E6 and E7, were confirmed by PCR amplification, and specific markers for corneal fibroblasts and epithelial cells were identified by RT–PCR and immunocytochemistry in both cell lines. Established human corneal fibroblasts (IHCF) and epithelial cell lines (IHCEp) were cultured on human AM with collagen scaffold and its specific gene expressions were identified by RT–PCR. Reconstructed 3D artificial cornea was sectioned, and expression patterns of specific markers were investigated by immunohistochemical staining. Results: Among the total independent clones, one of each cell lines cultured over 40 passages were chosen to further characterize the inherent biological properties. Infected oncoprotein was positively expressed in both IHCF and IHCEp. Differentiated epithelial cell marker K12 was expressed in both PHCEp and IHCEp, but not in PHCF and IHCF. Moreover, Cx43 was positively expressed in IHCEp, and vimentin, but not α–SM, was strongly expressed in IHCF. Immunohistochemical staining of reconstructed corneal sections exhibited fully differentiated p63 nuclear staining in basal cells, K3 over the epithelial layer, and linear collagen type IV under the basal cell layer. Furthermore, typical expression pattern of a strong TGF–ß1 staining over the entire epithelial layer was also detected. Conclusions: These results suggest that IHCF and IHCEp obtained here maintain normal corneal fibroblastic or epithelial characteristics, and reconstructed artificial cornea using human AM with collagen scaffold showed similar characteristics with normal human cornea. Therefore, it can be used not only for the biological studies on the human corneal fibroblasts and epithelial cells, but also for further applications such as the toxicological test. Acknowledgement: 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).
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