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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

The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and immortalized human corneal   endothelial cells (IHCEn). IHCEn were polygonal to slightly elongate in shape, similar to PHCEn.
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Related In: Results  -  Collection

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Figure 1: The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and immortalized human corneal endothelial cells (IHCEn). IHCEn were polygonal to slightly elongate in shape, similar to PHCEn.

Mentions: The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and IHCEn were observed using an inverted microspcope. IHCEn were polygonal to slightly elongated in shape, similar to PHCEn, but were siginificantly different from dendritic corneal fibroblasts and small globular-shaped epithelial cells (Fig. 1). Stable expression of E6/E7 mRNA was observed in IHCEn, but not in PHCEn by RT-PCR analysis. Messenger RNA of the corneal epithelial cell marker, keratin 12, was not expressed in PHCEn and IHCEn (Fig. 2).


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)

The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and immortalized human corneal   endothelial cells (IHCEn). IHCEn were polygonal to slightly elongate in shape, similar to PHCEn.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and immortalized human corneal endothelial cells (IHCEn). IHCEn were polygonal to slightly elongate in shape, similar to PHCEn.
Mentions: The morphological characteristics of isolated primary human corneal endothelial cells (PHCEn) and IHCEn were observed using an inverted microspcope. IHCEn were polygonal to slightly elongated in shape, similar to PHCEn, but were siginificantly different from dendritic corneal fibroblasts and small globular-shaped epithelial cells (Fig. 1). Stable expression of E6/E7 mRNA was observed in IHCEn, but not in PHCEn by RT-PCR analysis. Messenger RNA of the corneal epithelial cell marker, keratin 12, was not expressed in PHCEn and IHCEn (Fig. 2).

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