Korean J Ophthalmol.  2006 Mar;20(1):47-54. 10.3341/kjo.2006.20.1.47.

Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane

• Kim HJ
• Ryu YH
• Ahn JI
• Park JK
• Kim JC

• KMID: 1099072
• DOI: http://doi.org/10.3341/kjo.2006.20.1.47

Abstract

PURPOSE: To establish the immortalized human corneal endothelial cell line (IHCEn) by transducing human papilloma virus (HPV) 16 E6/E7 oncogenes, and to identify their characteristics when cultivated on a lyophilized human amniotic membrane (LAM). METHODS: Primary human corneal endothelial cells (PHCEn) were infected using a retroviral vector with HPV 16 E6/E7, and transformed cells were clonally selected by G418. Growth properties and characteristics of IHCEn were compared with PHCEn by cell counting and RT-PCR of VDAC3, SLC4A4, CLCN3, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn were cultured on LAM. Messenger RNA expressions of VDAC3, CLCN3, and Na+/K+ ATPase, and protein expressions of Na+/K+ ATPase and Col IV in IHCEn cultivated on LAM were investigated by RT-PCR, immunofluorescence, and immunohistochemical staining, respectively. RESULTS: Successful immortalization was confirmed by stable expression of HPV 16 E6/E7 mRNA by RT-PCR, and IHCEn exhibited typical corneal endothelial morphology. Doubling time of IHCEn was 30.15+/-10.96 hrs. Both IHCEn and PHCEn expressed VDAC3, CLCN3, SLC4A4, FGF-1, Col IV, and Na+/K+ ATPase. IHCEn cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish. Immunohistochemical staining and immunofluorescence revealed the positive expression of Na+/K+ ATPase and Col IV. CONCLUSIONS: IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

Keyword

Characterization; HPV 16 E6/E7; Immortalization; Immortalized human corneal endothelial cell lines; Lyophilized human amniotic membrane

MeSH Terms

Transfection
Reverse Transcriptase Polymerase Chain Reaction
Repressor Proteins/genetics/*pharmacology
RNA, Messenger/genetics
Protein-Tyrosine Kinases
Oncogene Proteins, Viral/genetics/*pharmacology
Immunohistochemistry
Humans
Gene Expression Regulation, Viral
Freeze Drying
Endothelium, Corneal/*cytology/drug effects/metabolism
Cell Line, Transformed
Cell Count
Amnion

Figure

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  Fig. 1 The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and immortalized human corneal endothelial cells (IHCEn). IHCEn were polygonal to slightly elongate in shape, similar to PHCEn.

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  Fig. 2 Messenger RNA expressions of E6/E7 and keratin 12 in PHCEn and IHCEn. Successful immortalization was confirmed by stable expressions of E6/E7 oncogenes only in IHCEn. Both PHCEn and IHCEn were confirmed as endothelial in origin by negative RT-PCR results for keratin12.

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  Fig. 3 Cell growth curve of established IHCEn. 1×105 IHCEn cells/ml were counted every two days for 14 days. A geometrical increase in cell number was detected from day seven on. Doubling time of IHCEn was 30.15±10.96 hrs.

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  Fig. 4 Messenger RNA expressions of several channel proteins. VDAC3 (voltage-dependent annion channel 3), SLC4A4 (sodium bicarbonate cotransporter, member 4), CLCN3 (chloride channel protein 3), FGF (fibroblast growth factor)-1, Col IV (collagen type IV), and Na+/K+ ATPase in PHCEn and IHCEn were examined by RT-PCR. M denotes the 1Kb ladder. There were no differences in expression pattern between PHCEn and IHCEn.

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  Fig. 5 Messenger RNA expressions of channel proteins and Na+/K+ ATPase in established IHCEn cultivated on a lyophilized amniotic membrane (LAM) and plastic culture dish (PL), respectively. IHCEn cultivated on LAM showed slightly stronger expressions of all factors.

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  Fig. 6 Immunohistochemical staining of Col IV and Na+/K+ ATPase in IHCEn cultivated on LAM. (A) H&E staining, (B) immunohistochemical staining of Col IV, and (C) immunofluorescence of Na+/K+ ATPase. Established cell lines cultivated on LAM showed positive expressions of Col IV (arrow head) and Na+/K+ (green).

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