Characterization of immortalized human corneal endothelial cell line using HPV 16 E6/E7 on lyophilized human amniotic membrane.
OBJECTIVE
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.
Kim
Hyun-Ju
HJ
Department of Ophthalmology, Chung-Ang University Yongsan Hospital, Seoul, Korea.
Ryu
Yang-Hwan
YH
Ahn
Jae-Il
JI
Park
Jeong-Keuk
JK
Kim
Jae-Chan
JC
D000650:Amnion; D002452:Cell Count; D002461:Cell Line, Transformed; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D005612:Freeze Drying; D015967:Gene Expression Regulation, Viral; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases; D012333:RNA, Messenger / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D012097:Repressor Proteins; D011505:Protein-Tyrosine Kinases