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Characterization of immortalized human corneal endothelial cell line using HPV 16 E6/E7 on lyophilized human amniotic membrane.

Kim HJ, Ryu YH, Ahn JI, Park JK, Kim JC - Korean J Ophthalmol (2006)

Bottom Line: 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 cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish.IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Chung-Ang University Yongsan Hospital, Seoul, Korea.

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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Related in: MedlinePlus

Cell growth curve of established IHCEn. 1×105 IHCEn cells/ml were counted every two days for 14 days. A geometrical increase in cell number was detected from day seven on. Doubling time of IHCEn was 30.15±10.96 hrs.
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Figure 3: Cell growth curve of established IHCEn. 1×105 IHCEn cells/ml were counted every two days for 14 days. A geometrical increase in cell number was detected from day seven on. Doubling time of IHCEn was 30.15±10.96 hrs.

Mentions: Proliferative characteristics of IHCEn were elucidated by cell counting every two days for 14 days. The cell growth curve of IHCEn showed typical S-curves: minimal growth for the initial seven days, geometrical growth until day 14, and decreased cell numbers after 14 days. The estimated cell doubling time of IHCEn was 30.15±10.96 hrs (Fig. 3). IHCEn showed a more extended life span (over passage 30), and more rapid proliferation than PHCEn (data not shown). Transformed traits, except for the transducing of HPV 16 E6/E7 oncogenes, were examined by RT-PCR of several channel proteins including voltage-dependent annion channel 3 (CDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), fibroblast growth factor (FGF)-1, type IV collagen (Col IV), and Na+/K+ ATPase in PHCEn and IHCEn. mRNAs for all of the mentioned proteins were expressed in IHCEn, with similar expression patterns observed in PHCEn.


Characterization of immortalized human corneal endothelial cell line using HPV 16 E6/E7 on lyophilized human amniotic membrane.

Kim HJ, Ryu YH, Ahn JI, Park JK, Kim JC - Korean J Ophthalmol (2006)

Cell growth curve of established IHCEn. 1×105 IHCEn cells/ml were counted every two days for 14 days. A geometrical increase in cell number was detected from day seven on. Doubling time of IHCEn was 30.15±10.96 hrs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2908816&req=5

Figure 3: Cell growth curve of established IHCEn. 1×105 IHCEn cells/ml were counted every two days for 14 days. A geometrical increase in cell number was detected from day seven on. Doubling time of IHCEn was 30.15±10.96 hrs.
Mentions: Proliferative characteristics of IHCEn were elucidated by cell counting every two days for 14 days. The cell growth curve of IHCEn showed typical S-curves: minimal growth for the initial seven days, geometrical growth until day 14, and decreased cell numbers after 14 days. The estimated cell doubling time of IHCEn was 30.15±10.96 hrs (Fig. 3). IHCEn showed a more extended life span (over passage 30), and more rapid proliferation than PHCEn (data not shown). Transformed traits, except for the transducing of HPV 16 E6/E7 oncogenes, were examined by RT-PCR of several channel proteins including voltage-dependent annion channel 3 (CDAC3), sodium bicarbonate cotransporter member 4 (SLC4A4), chloride channel protein 3 (CLCN3), fibroblast growth factor (FGF)-1, type IV collagen (Col IV), and Na+/K+ ATPase in PHCEn and IHCEn. mRNAs for all of the mentioned proteins were expressed in IHCEn, with similar expression patterns observed in PHCEn.

Bottom Line: 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 cultivated on LAM showed stronger expression of VDAC3, CLCN4, and Na+/K+ ATPase mRNA than on plastic culture dish.IHCEn were successfully established, and LAM is a good substrate for the culture of human corneal endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Chung-Ang University Yongsan Hospital, Seoul, Korea.

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.

Show MeSH
Related in: MedlinePlus