각막내피세포에서 Ceramide에 의해 유발된 Apoptosis

Abstract

Purpose: To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. Methods: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 M of ceramide type Ⅱ, Ⅵ and phytoceramide type Ⅱ, Ⅵ. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin Ⅴ for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK), specific caspase-8 inhibitor (IETD-CHO) and specific caspase-9 inhibitor (Z-VAD-FMK), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide. Results: Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody. Conclusions: Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.