Proliferation of Corneal Endothelial Cells by Delivery of SV40 Large T Antigen

PURPOSE: To determine whether the delivery of the SV40 large T-antigen is a feasible method for transiently inducing proliferation of corneal endothelial cells, we delivered liposome-protein complex into bovine corneal endothelial cells(BCEC). METHOD: SV40 large T-antigen protein was introduced into BCEC and positive cells were identified by immunohistochemistry. Quiescent BCECs were double-labeled using BrdU as a measure of de novo DNA synthesis and the Ki-67 was detected by standard immunohistochemical methods. RESULT: The treatment of quiescent BCECs with large T antigen caused an increase in BrdU incorporation and Ki-67 expression. It was tested by time-course study. CONCLUSION: This finding suggests that liposome-mediated delivery of transforming proteins could be a method to transiently induce corneal endothelial cell proliferation.

Transfection of SV 40 Large T Antigen into Corneal Endothelial Cells

The coeneal endothelium is essential for the maintenance of normal corneal hydration, thickness, and transparency. However, corneal endothelial cells are incapable of significant proliferation in vivo. As we age, the density of corneal endothelial (CEN) cells gradually decreases. The goal of our study is to explore the possibility of enhancing the proliferation of corneal endothelial cells by introduction of SV 40 large T antigen, a transforming protein. To this end, introduction of protein into CEN cells was assessed by liposome assisted beta-galactosidase transfection in vivo, ex vivo, and in vivo. In all cases, cells treated with liposome-protein complex have shown dramatic blue stain in beta-galactosidase activity staining. This result convinced us that we could artificially introduce a foreign protein into a cell. To ascertain where SV 40 large T antigen is localized in the cell, purified SV 40 large T antigen was transfected into the cells using liposome and its presence was determined immunohistochemically. We show that the liposome delivered SV 40 large is localized in the nucleus and mitotic figures which may suggest its functional activity.

Phototoxicity of Riboflavin and Rose bengal on Cultured Bovine Lens Epithelial Cells in vitro

Light may induce various kindsof oxygen free radicals in the eye and show a toxic effect on lens epithelial cells (LEC)as well as cataract formation. In this study, we examined the oxygen free radical injury of LEC using photosensitizer like riboflavin (RF) or rese bengal (RB) and blocking effect of antagonists on photoxicity of LEC. We cultured bovine lens epithelial cells (BLEC) to 24 well plate at the concentration of 5*10 in each well. BLEC were exposed with a illumination of 1200 lux for 2 hours under th concentration of 10, 25, 50, 75 and 100 uM of RF and 5, 10, 15, and 20 uM of RB, respectively. The pattern of BLEC death was observed with inverted light microscope and the quantitation of BLEC cytotoxicity was calculated using 3-(4, 6-dimethylthiazol-2-yl) -2, 5-diphenyl tetrazolium bromide(MTT) assay. The blocking effects of catalase, sodium azide, vitamin C, and vitamin E on BLEC phototoxicity were also evaluated. The concentration of RF inducing a prominent phototoxicity with a light of 1200 lux for 2 hours was determined to 50uM and that of RB was determined to 10uM(p<0.05). The quantiative cytotoxicity of BLEC was prominent at 24 hours dark adaptation following 2 hours illumination. In the pattern of phototoxicity of BLEfC, RF induced a cell death showing shrinkage of cell membrane and arborization of process, while RB induced a cell death showing swelling of cytoplasm. In the experiment of the scavengers` effects on phototoxicity of BLEC, catalase (3.75 U/ml) attenuated selectively a phototoxicity of BLEC induced by RF and sodium azide (imM) attenuated selectively a phototoxicity induced by RB. Conclusively, we could find hydrogen peroxide and singlet oxygen were main oxygen free radicals induced by RF and RB, respectively and the patterns of cell death induced by photosensitizer of RF and RB were cellular shrinkage and swelling, repectively.