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목적 : To examine the role of apoptosis in cultured human primary corneal epithelial cells (pHCECs) exposed high-glucose environment and, to evaluate the effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the apoptotic response in pHCECs exposed high-glucose environment.
방법 : pHCECs were exposed to glucose (0, 5, 10, 25 mM). To assess intracellular ROS, fluorescent microscope using DCFH-DA fluorescence was performed. To assess apoptosis, flowcytometric analysis was performed and levels of Bcl-xL, phosphorylated JNK (pJNK) were detected using Western blotting at pHCECs exposed each concentration of glucose with or without recombinant human NGF (rhNGF) and EGF (rhEGF). To confirm restricted migration, scratched-induced directional wounding assay, F-actin staining, and levels of Paxillin, integrin-β2 were detected using western blotting.
결과 : A high-glucose environment enhanced production of ROS, reduced cell viability, increased number of apoptotic cells, increased expression of pJNK and reduced expression of Bcl-xL in pHCECs in a dose dependent manner. Addition of rhNGF and rhEGF decreased the number of apoptotic cells, reduced the expression of pJNK and increased the expression of Bcl-xL. In addition, a high-glucose environment suppressed epithelial cell migration and, F-actin was decreased in quality or quantity. The expression of Paxillin and integrin-β were reduced in high-glucose environment.
결론 : A high-glucose environment enhanced apoptosis of pHCECs by activating JNK sinaling and contributed to impaired epithelial wound healing. And suppressed epithelial cell migration was also contributed to impaired epithelial wound healing. Increased levels of NGF and EGF may contribute to the reduced apoptosis of pHCECs. Targeting apoptosis may present novel therapeutic approaches to manage diabetic keratopathy.
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