Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane.
- Author:
Hyun Ju KIM
1
;
Yang Hwan RYU
;
Jae Il AHN
;
Jeong Keuk PARK
;
Jae Chan KIM
Author Information
- Publication Type:Original Article ; Comparative Study ; In Vitro ; Research Support, Non-U.S. Gov't
- Keywords: Characterization; HPV 16 E6/E7; Immortalization; Immortalized human corneal endothelial cell lines; Lyophilized human amniotic membrane
- MeSH: 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
- From:Korean Journal of Ophthalmology 2006;20(1):47-54
- CountryRepublic of Korea
- Language:English
- 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.
