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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

Messenger RNA expressions of several channel proteins. VDAC3 (voltage-dependent annion channel 3), SLC4A4 (sodium bicarbonate cotransporter, member 4), CLCN3 (chloride channel protein 3), FGF (fibroblast growth factor)-1, Col IV (collagen type IV), and Na+/K+ ATPase in PHCEn and IHCEn were examined by RT-PCR. M denotes the 1Kb ladder. There were no differences in expression pattern between PHCEn and IHCEn.
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Figure 4: Messenger RNA expressions of several channel proteins. VDAC3 (voltage-dependent annion channel 3), SLC4A4 (sodium bicarbonate cotransporter, member 4), CLCN3 (chloride channel protein 3), FGF (fibroblast growth factor)-1, Col IV (collagen type IV), and Na+/K+ ATPase in PHCEn and IHCEn were examined by RT-PCR. M denotes the 1Kb ladder. There were no differences in expression pattern between PHCEn and IHCEn.

Mentions: Introduction of E6/E7 oncogenes facilitates the degradation of p53 and progression into the S phase synergistically.29-32 Moreover, their stability was also confirmed by observing which gene was non-carcinogenic when injected into nude mice.33 Therefore, we were able to successfully obtain abundant populations of highly proliferative and stably immortalized human corneal endothelial cells. Furthermore, it was also identified that established IHCEn maintained normal corneal endothelial functions similar to PHCEn by confirming VDAC3, CLCN3, CLC4A4, and Na+/K+ ATPase mRNA expressions (Fig. 4).


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)

Messenger RNA expressions of several channel proteins. VDAC3 (voltage-dependent annion channel 3), SLC4A4 (sodium bicarbonate cotransporter, member 4), CLCN3 (chloride channel protein 3), FGF (fibroblast growth factor)-1, Col IV (collagen type IV), and Na+/K+ ATPase in PHCEn and IHCEn were examined by RT-PCR. M denotes the 1Kb ladder. There were no differences in expression pattern between PHCEn and IHCEn.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Messenger RNA expressions of several channel proteins. VDAC3 (voltage-dependent annion channel 3), SLC4A4 (sodium bicarbonate cotransporter, member 4), CLCN3 (chloride channel protein 3), FGF (fibroblast growth factor)-1, Col IV (collagen type IV), and Na+/K+ ATPase in PHCEn and IHCEn were examined by RT-PCR. M denotes the 1Kb ladder. There were no differences in expression pattern between PHCEn and IHCEn.
Mentions: Introduction of E6/E7 oncogenes facilitates the degradation of p53 and progression into the S phase synergistically.29-32 Moreover, their stability was also confirmed by observing which gene was non-carcinogenic when injected into nude mice.33 Therefore, we were able to successfully obtain abundant populations of highly proliferative and stably immortalized human corneal endothelial cells. Furthermore, it was also identified that established IHCEn maintained normal corneal endothelial functions similar to PHCEn by confirming VDAC3, CLCN3, CLC4A4, and Na+/K+ ATPase mRNA expressions (Fig. 4).

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