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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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Immunohistochemical staining of Col IV and Na+/K+ ATPase in IHCEn cultivated on LAM. (A) H&E staining, (B) immunohistochemical staining of Col IV, and (C) immunofluorescence of Na+/K+ ATPase. Established cell lines cultivated on LAM showed positive expressions of Col IV (arrow head) and Na+/K+ (green).
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Figure 6: Immunohistochemical staining of Col IV and Na+/K+ ATPase in IHCEn cultivated on LAM. (A) H&E staining, (B) immunohistochemical staining of Col IV, and (C) immunofluorescence of Na+/K+ ATPase. Established cell lines cultivated on LAM showed positive expressions of Col IV (arrow head) and Na+/K+ (green).

Mentions: In order to elucidate the efficiency of LAM for the human corneal endothelial niche, IHCEn were cultured on LAM and harvested for characterization. Messenger RNA expressions of several channel proteins and Na+/K+ ATPase were observed in established IHCEn cultivated on LAM. Moreover, their expressions were stronger than in cells grown on a plastic culture dish (Fig. 5). Immunohitochemical staining of Col IV and immunofluorescence of Na+/K+ ATPase in IHCEn cultivated on LAM also showed positive expressions results similar to RT-PCR (Fig. 6).


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)

Immunohistochemical staining of Col IV and Na+/K+ ATPase in IHCEn cultivated on LAM. (A) H&E staining, (B) immunohistochemical staining of Col IV, and (C) immunofluorescence of Na+/K+ ATPase. Established cell lines cultivated on LAM showed positive expressions of Col IV (arrow head) and Na+/K+ (green).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Immunohistochemical staining of Col IV and Na+/K+ ATPase in IHCEn cultivated on LAM. (A) H&E staining, (B) immunohistochemical staining of Col IV, and (C) immunofluorescence of Na+/K+ ATPase. Established cell lines cultivated on LAM showed positive expressions of Col IV (arrow head) and Na+/K+ (green).
Mentions: In order to elucidate the efficiency of LAM for the human corneal endothelial niche, IHCEn were cultured on LAM and harvested for characterization. Messenger RNA expressions of several channel proteins and Na+/K+ ATPase were observed in established IHCEn cultivated on LAM. Moreover, their expressions were stronger than in cells grown on a plastic culture dish (Fig. 5). Immunohitochemical staining of Col IV and immunofluorescence of Na+/K+ ATPase in IHCEn cultivated on LAM also showed positive expressions results similar to RT-PCR (Fig. 6).

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