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目的明确OGD损伤诱导的PC12细胞激活素A(Activin A,ActA)信号在细胞内基于Smad4的转导模式及作用。方法使用鼠神经生长因子(NGF,50ng/ml)诱导大鼠嗜铬细胞瘤PC12细胞向神经元样转化,利用无糖DMEM培养液联合连二亚硫酸钠(1mmol/L)构建神经元氧糖剥夺(oxygen-glucose deprivation,OGD)模型,体外模拟神经元缺血性损伤。OGD 0、3h、6h、12h后Realtime-PCR检测ActA mRNA表达水平;激光共聚焦观察免疫荧光染色后的Smad4质核分布情况。结果 NGF诱导后PC12细胞贴壁并伸出较长的突触,向神经元样转化。细胞OGD处理3h后ActA mRNA表达水平较0h显著升高,6h开始下降;OGD3h后Smad4在细胞核内的分布较OGD 0h明显增加,6小时开始下降。结论 OGD损伤可诱导神经元样PC12细胞ActA信号激活,Smad4可通过质核移位介导ActA信号在受体水平下游的信号转导,且随时间延长呈动态变化。
OBJECTIVE: To determine Smad4-based transduction patterns and roles of Activin A (ActA) signaling in PC12 cells induced by OGD injury. Methods Neuronal transformation of PC12 cells from rat pheochromocytoma was induced by using rat nerve growth factor (NGF, 50ng / ml). Glucose deprivation in neurons was induced by glucose - free DMEM combined with sodium dithionite (1mmol / L) oxygen-glucose deprivation (OGD) model to simulate neuronal ischemic injury in vitro. Real-time PCR was used to detect the expression of ActA mRNA at 0, 3h, 6h and 12h after OGD. The distribution of Smad4 nucleus after immunofluorescence staining was observed by laser scanning confocal microscope. Results After NGF induction, PC12 cells adherent and extended longer synapses to neuron-like transformation. After 3 h OGD treatment, the expression of ActA mRNA increased significantly from 0 h to 6 h and then decreased. The distribution of Smad4 in OGD 3 h after OGD increased obviously at 0 h and decreased at 6 h. Conclusion OGD can induce ActA signal activation in neuron - like PC12 cells. Smad4 mediates the signal transduction of ActA signal downstream of receptor by nuclear translocation, and it changes dynamically with time.