钙拮抗剂改善铁诱导的小鼠神经干细胞系C17.2细胞凋亡  

Calcium channel blockers alleviated neural stem cell line C17.2 cells apoptosis induced by iron

作　　者：陈文雄 黄珍妮[2] 杨默[3] 吴武田[4] 苏焕兴[5] 陈志峰[2] 

机构地区：[1]广州市妇女儿童医疗中心脑病中心神经内科,广东广州510623 [2]香港大学李嘉诚医学院儿童与青少年系 [3]南方医科大学南方医院儿科 [4]香港大学李嘉诚医学院解剖学系 [5]澳门大学中华医药研究院中药质量研究国家重点实验室

出　　处：《中华高血压杂志》2016年第8期763-773,共11页Chinese Journal of Hypertension

基　　金：香港大学CRCG-HKU研究基金(10207982-08296-21100-323)

摘　　要：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 Excessive iron accumulation in the brain with oxidative damage has been found in neurode- generative disorders and hemorrhagic stroke. The treatment of the above disorders with neural stem cells （NSCs） is a promising intervention strategy. To investigate the effects of iron overload on NSCs and explore its protection strategies is an urgent problem to be solved. Calcium channel acts as an alternative channel for iron overload in the neural cell, and hypothetically its blocker can be used to alleviate the toxic effect of iron overload on NSCs. Objective To investigate the effects of iron-overload on cell viability, apoptosis and extracellular signal-regulated kinases （ERK） phosphorylation of mouse neural stem cell line C17.2 cells, and to explore the protective potential of calcium channel blockers on iron-injured C17.2 cells. Methods Mouse neural stem cell line C17.2 cells were used as cell models. Cell activity was deicected by XTT colorimetric assay. The apoptotic cells were measured by Annexin V/propidium iodide（PI）, activated caspase-3 and mitochondrial membrane potential （JC-1）, respectively, and the phosphorylation level of ERK detected by anti-phospho-ERK1/2 antibody with flow cytometry. Results Iron overload significantly decreased C17.2 cell viability in a dose（0.15-1.80 mmol/L） and time（24- 48 h）-dependent manner. Furthermore, Iron overload significantly induced cell apoptosis, activated caspase-3, and caused mitochon- drial damage of C17.2 cells. Calcium channel blockers （flunarizine and nimodipine） significantly alleviated apoptosis, decreased caspase-3 activation, ameliorated mitochondrial damage, and increased C17.2 cells viability induced by 0.60 mmol/L iron trichloride. Additionally, iron overload （0.60 mmol/L） induced ERK phosphorylation of C17.2 cells, which could be obviously inhibited by calcium channel blockers （flunarizine and nimodipine）. Conclusion ERK phosphorylation of mouse neural stem cell line C17.2 can be induced by iron overload, which

关 键 词：铁超负荷 神经干细胞 钙拮抗剂 细胞外信号调节激酶 

分 类 号：R965[医药卫生—药理学]