Characterization of immortalized human corneal endothelial cell line using HPV 16 E6/E7 on lyophilized human amniotic membrane.
- Authors
- Kim, H.J.; Ryu, Y.H.; Ahn, J.I.; Park, J.K.; Kim, J.C.
- Issue Date
- Mar-2006
- Publisher
- 대한안과학회
- Keywords
- Characterization; HPV 16 E6/E7; Immortalization; Immortalized human corneal endothelial cell lines; Lyophilized human amniotic membrane; Characterization; HPV 16 E6/E7; Immortalization; Immortalized human corneal endothelial cell lines; Lyophilized human amniotic membrane
- Citation
- Korean journal of ophthalmology : KJO, v.20, no.1, pp 47 - 54
- Pages
- 8
- Journal Title
- Korean journal of ophthalmology : KJO
- Volume
- 20
- Number
- 1
- Start Page
- 47
- End Page
- 54
- URI
- https://scholarworks.bwise.kr/cau/handle/2019.sw.cau/47061
- DOI
- 10.3341/kjo.2006.20.1.47
- ISSN
- 1011-8942
2092-9382
- 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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