| Summary: | 碩士 === 台北醫學院 === 醫學研究所 === 88 === The redox signaling has been shown to correlate with various biological processes including cell proliferation, differentiation and apoptosis. In our research, the rat adrenal pheochromocytoma (PC12) cells was used as a cell model to investigate the roles of reactive oxygen species (ROS) and antioxidant enzymes in nerve growth factor (NGF)-induced differentiation. The neuronal specific differentiation markers, tyrosine hydroxylase (TH) and neurofilament (NF)-L, were detected by immuno- blot to confirm the differentiation stage of PC12 cells. Among numerous antioxidant compounds or enzymes, only N-acetylcysteine (NAC) could suppress the NGF-induced PC12 differentiation. However, tiron (.O2- specific scavenger) and mannitol (.OH specific scavenger) exerted only minor effects. By flowcytometry method, minor but significant generation of cellular hydrogen peroxide (H2O2), but not superoxide anion (.O2-), was detected after treatment with NGF. These results indicated that H2O2 might play a pivotal role during PC12 differentiation. The antioxidant enzymes might be responded to redox homeostasis during PC12 differentiation. By enzyme kinetic analysis, the activities of catalase (CAT)、glutathione reductase (GRx)、copper-zinc superoxide dismutase (Cu/ZnSOD) and manganese superoxide dismutase (MnSOD) were examined. Only the activity of CAT was elevated slightly but significantly (1.5 folds) after NGF-induced differentiation of PC12 cells.
ROS has been suggested as a signaling molecule to regulate the so-called ROS-sensitive MAPKs, such as extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK) and p38. Therefore, the activities of ROS-sensitive MAPKs were analyzed by immuno-blot. ERK and JNK were activated dramatically during the initial stage of NGF-induced PC12 differentiation as prolonged and transient profiles, respectively. Nevertheless, p38 activity was decreased (~80 %) at 20 minutes time-point and then recovered to basal level. After pre-treatment with NAC, the NGF-induced activation of ERK was suppressed, which suggested that ERK might be a down-stream molecule of H2O2 production mediated by NGF. Based on these results, ROS, especially H2O2, might play a pivotal role during NGF-induced differentiation of PC12 cells.
|
|---|
