Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane
Korean Journal of Ophthalmology
; : 47-54, 2006.
Article
em En
| WPRIM
| ID: wpr-72710
Biblioteca responsável:
WPRO
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.
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Texto completo:
1
Índice:
WPRIM
Assunto principal:
Proteínas Repressoras
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Proteínas Tirosina Quinases
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RNA Mensageiro
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Linhagem Celular Transformada
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Endotélio Corneano
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Imuno-Histoquímica
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Transfecção
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Regulação Viral da Expressão Gênica
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Contagem de Células
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Proteínas Oncogênicas Virais
Tipo de estudo:
Prognostic_studies
Limite:
Humans
Idioma:
En
Revista:
Korean Journal of Ophthalmology
Ano de publicação:
2006
Tipo de documento:
Article