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.
Subject(s)
Humans , Transfection , Reverse Transcriptase Polymerase Chain Reaction , Repressor Proteins/genetics , RNA, Messenger/genetics , Protein-Tyrosine Kinases , Oncogene Proteins, Viral/genetics , Immunohistochemistry , Gene Expression Regulation, Viral , Freeze Drying , Endothelium, Corneal/cytology , Cell Line, Transformed , Cell Count , AmnionABSTRACT
Tested serum of donors from the Blood Bank unit of Siriraj Hospital was collected. Only negative hepatitis B-antigen determined by RPHA (reverse passive haemagglutination) method was selected. The sera were pooled as one pooled serum. Filtrations were performed after adjusting some biochemical constituents. To obtain lyophilized serum, the pooled serum was subjected to freeze-dry process. It was divided into aliquot of 5.0 ml in each vial, then quick freezed and dried in the lyophilizer. Precision of between-vial filling volume was studied. We found that the coefficient if variation was 0.01%. Reconstitution of the lyophilized serum before use was performed by adding 5.0 ml of distilled water to each vial. The coefficient variation of between-vial biochemical constituents was found to be less than 0.5%. The precision of 19 routine biochemical tests in these Home-Made lyophilized human serum were compared to those of 3 batches of well-known commercial control materials (ORTHO, MONITROL, VALIDATE). By F-test analysis, the coefficient variation of most biochemical tests in the Home-Made lyophilized human serum was not significant different from those of the commercial ones (P>0.05). The long-term consistency of the biochemical constituents was studied. In a duration of one year, from March 1985 to February 1986, three-month periods means and standard deviations were analyzed. The results showed no significant difference for these period of study (P>0.05). Since 1985, the Home-Made lyophilized serum has been used as internal control material in our Central Clinical Chemistry Laboratory, Faculty of Medicine Technology, Mahidol University in place of imported control serum. The external control evaluation was successfully reported by the International External Quality Control Assessment Scheme. (Birmingham UK) and the improvement of Overall Mean Running Variance Index Score (OMRVIS) is obviously seen in the graphic curve.