배양된 망막신경절세포주에서 허혈전처치와 Protein kinase C의 역할

Ischemic Preconditioning and the Role of Protein Kinase C in Cultured Retinal Ganglion Cell Line

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 μM 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 α, β, γ, δ, ε, ζ 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 μM 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 α and ε 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 α and ε suggest some functional relevance of these isoenzymes to the cytoprotective effects conferred by hypoxic preconditioning. J Korean Ophthalmol Soc 49(6):979-986, 2008