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背景脑中铁超负荷诱导氧化损伤见于出血性脑卒中和神经退行性障碍,神经干细胞是治疗这种障碍的充满前景的干预策略。铁超负荷对干细胞影响及探寻保护策略是亟待解决的问题。钙通道扮演铁超负荷时进入神经细胞的替代通道,推测钙拮抗剂可改善铁超负荷对神经干细胞的毒性作用。目的探讨铁超负荷对小鼠神经干细胞系C17.2细胞的细胞活性、凋亡及细胞外信号调节激酶(ERK)磷酸化的影响;研究钙拮抗剂对铁诱导C17.2细胞损伤是否具有保护作用。方法以小鼠神经干细胞系C17.2细胞为细胞模型。XTT比色法检测细胞活性;Annexin V/碘化丙啶(PI),活化半胱氨酸天冬氨酸蛋白酶(caspase)-3及线粒体膜电位(JC-1)流式细胞仪检测细胞凋亡;抗磷酸化-ERK1/2流式细胞仪检测ERK磷酸化水平。结果铁超负荷呈浓度(0.15~1.80mmol/L)及时间(24~48h)依赖趋势减少C17.2细胞活性,诱导细胞凋亡,caspase-3活化及线粒体损伤;钙拮抗剂(氟桂利嗪或尼莫地平)可以显著改善0.60mmol/L三氯化铁超负荷诱导的C17.2细胞早期凋亡细胞百分数及总死亡细胞百分数,减少caspase-3活化及线粒体损伤,增加细胞活性(均P<0.05)。0.60mmol/L超负荷铁可以显著诱导C17.2细胞ERK磷酸化,钙拮抗剂(氟桂利嗪或尼莫地平)显著抑制铁超负荷所诱导的C17.2细胞ERK磷酸化。结论铁超负荷可诱导小鼠神经干细胞系C17.2细胞ERK磷酸化,导致线粒体介导的凋亡。钙拮抗剂可抑制上述过程,改善铁诱导的C17.2细胞凋亡。
BACKGROUND OF THE INVENTION Iron-overload induced oxidative damage in the brain is found in hemorrhagic stroke and neurodegenerative disorders. Neural stem cells are a promising intervention strategy for the treatment of this disorder. Iron overload on the impact of stem cells and explore protection strategies is an urgent problem to be solved. Calcium channels play an alternative channel into nerve cells during overload of iron, suggesting that calcium antagonists can improve the toxic effects of iron overload on neural stem cells. Objective To investigate the effects of iron overload on cell viability, apoptosis and extracellular signal-regulated kinase (ERK) phosphorylation in mouse neural stem cell line C17.2 cells and to investigate whether calcium antagonists have protective effects on iron-induced C17.2 cell injury effect. Methods The mouse neural stem cell line C17.2 cells were used as the cell model. Cell viability was detected by XTT assay. Annexin V / propidium iodide (PI), caspase-3 and mitochondrial membrane potential (JC-1) Anti-phosphorylation-ERK1 / 2 flow cytometry was used to detect ERK phosphorylation. Results Iron overload increased the cell viability and induced apoptosis, caspase-3 activation and mitochondrial damage in a concentration dependent manner (0.15-1.80 mmol / L) and time (24-48 h). Calcium antagonists Oxazine or nimodipine) could significantly improve the percentage of early apoptotic cells and total dead cells of C17.2 cells induced by 0.60mmol / L ferric chloride overload, decrease the activation of caspase-3 and mitochondrial damage and increase the activity of cells P <0.05). 0.60 mmol / L iron overload significantly induced ERK phosphorylation in C17.2 cells, and calcium antagonist (flunarizine or nimodipine) significantly inhibited ERK phosphorylation induced by iron overload in C17.2 cells. Conclusion Iron overload can induce phosphorylation of ERK in mouse neural stem cell line C17.2, resulting in mitochondria-mediated apoptosis. Calcium antagonists can inhibit the above process, improve iron-induced apoptosis of C17.2 cells.