배양된 망막신경절세포주에서 허혈전처치와 Protein kinase C의 역할

Abstract

PURPOSE: To investigate the cellular protective effects of hypoxic preconditioning against oxidative stress in a staurosporine-differentiated RGC-5 cell line and the relevance of protein kinase C subtype expression. METHODS: The minimum staurosporine concentration and exposure time necessary to morphologically fully differentiate RGC-5 cells were determined. Cytotoxic injury was provided by oxidative stress with 800 micrometer hydrogen peroxide (H2O2) for 15 hours to morphologically fully-differentiated cells. The cytoprotective effect of hypoxic preconditioning was found by exposing the cell line to 0.3% oxygen for different periods of time. Quantifiable changes in the expression of mRNAs and proteins of the isoenzymes alpha, beta, gamma, delta, epsilon, zeta of protein kinase C were determined before and after 1, 2, 15, and 24 hours of hypoxic preconditioning. RESULTS: Axonal growth in RGC-5 cells after the induction of differentiation with staurosporine caused these cells to resemble neurons. The minimal concentration and exposure time to staurosporine that evoked full differentiation of RGC-5 cells was exposure to 2 micrometer staurosporine for 1 hour. An LDH assay demonstrated that hypoxic preconditioning had neuroprotective effects against hydrogen peroxide-induced oxidative stress. Protein and mRNA levels of PKC isoforms alpha and epsilon increased after preconditioning. CONCLUSIONS: Hypoxic preconditioning of staurosporine-differentiated RGC-5 cells had a cytoprotective effect against oxidative stress. The associated increase of mRNA and proteins of PKC isoenzymes alpha and epsilon suggest some functional relevance of these isoenzymes to the cytoprotective effects conferred by hypoxic preconditioning