| 摘要: | 氧化還原狀態(redox status)與許多細胞生理過程息息相關，包括細胞增生、分化及凋亡。本論文以大鼠腎上腺的嗜鉻細胞瘤細胞(PC12)為細胞模式進行系列研究，探討神經生長因子(nerve growth factor，NGF)誘導PC12細胞分化的過程中，活性氧分子(reactive oxygen species，ROS)與抗氧化酵素(antioxidant enzymes)所扮演的角色。
以神經突觸外生(neurite outgrowth)及神經細胞特有的指標蛋白包括：tyrosine hydroxylase (TH)與neurofilament-L (NF-L)的表現，確定NGF可誘導PC12分化。為瞭解此過程中是否有ROS的參與，因此利用多種抗氧化物或抗氧化酵素處理細胞。結果發現，NAC可有效抑制NGF誘導PC12分化，但‧OH的特異性清除劑mannitol以及‧O2-的特異性清除劑tiron則無明顯地抑制作用。利用流式細胞儀的技術，以2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA)及dihydroethidium (HEt)分別檢測H2O2及.O2-，發現在NGF處理1分鐘時胞內H2O2約提升3倍左右，但.O2-並無明顯變化情形。由上述結果顯示，H2O2可能參與NGF誘導PC12的細胞分化過程並具備要能。此外，由於H2O2的變化可能改變胞內抗氧化酵素的活性，因此本論文進一步分析在PC12分化過程中，抗氧化酵素包括catalase (CAT)、glutathione reductase (GRx)、copper-zinc superoxide dismutase (Cu/ZnSOD)及manganese superoxide dismutase (MnSOD)活性的變化，發現僅有CAT的活性在分化中期有明顯增加1.5倍的現象(p<0.01)。
Mitogen-activated protein kinase (MAPKs)為胞內重要的訊息傳遞分子之一，其中extracellular signal-regulated protein kinase (ERK)、c-Jun N-terminal protein kinase (JNK)與p38之活性，易受到ROS之調控。本論文分析在NGF誘導PC12細胞分化過程中，這些ROS敏感性MAPKs的表現情形，及其活性是否受到ROS的調節。結果發現，ERK在經過NGF處理5分鐘時即有8.7倍的活化，並能保持2~3倍的活化程度至3小時後；JNK則在10分鐘時有短暫地活化14.2倍，而40~60分鐘時有第二次較弱的活化，之後便回復至基礎值(basal level)；相反的，p38在20分鐘時活性會降低80 %，之後會漸漸恢復至基礎值左右。在經過NAC的前處理後，NGF誘導ERK的活化現象，幾乎可被完全抑制，表示ERK的活化可能經由NGF誘導產生的H2O2所調控。綜合上述結果顯示，NGF可能藉由提升胞內H2O2濃度，並透過ROS敏感性MAPKs的作用，最後導致PC12神經細胞的分化。但其詳細機轉，仍有待進一步確認。
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. |
