학술논문
Characterization of Immortalized Human Corneal Endothelial Cell Line using HPV 16 E6/E7 on Lyophilized Human Amniotic Membrane
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- 영문명
- 발행기관
- 대한안과학회
- 저자명
- Hyun-Ju Kim Yang-Hwan Ryu Jae-Il Ahn Jeong-Keuk Park Jae-Chan Kim
- 간행물 정보
- 『The Korean Journal of Ophthalmology』Vol.20 No.1, 47~54쪽, 전체 8쪽
- 주제분류
- 의약학 > 기타의약학
- 파일형태
- 발행일자
- 2006.03.30
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국문 초록
영문 초록
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. Korean Journal of Ophthalmology 20(1):47-54, 2006
목차
Materials and Methods
Results
Discussion
키워드
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