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目的探讨电磁脉冲(EMP)致体外培养的海马神经元损伤的机制及药物防护的可能性。方法EMP的照射条件为6×104V·m-1,脉冲上升时间为20ns,脉宽为30μs,频率为2.5脉冲·min-1,作用时间为2min。原代培养的海马神经元经过MK801和尼非地平预处理后进行EMP照射,采用MTT法对细胞活力进行测定;FACS法检测细胞凋亡率;Fluo-3-AM荧光探针负载、激光共聚焦显微镜扫描测定海马神经元胞内游离钙离子〔Ca2+〕i浓度。结果MK801组和MK801+尼非地平组反应细胞增殖活力的吸光度分别为0.25±0.06和0.27±0.07,明显高于单纯EMP照射组(0.17±0.08,P<0.05);MK801+尼非地平组的细胞凋亡率(53.69±13.60)%较单纯EMP组(68.63±9.04)%明显下降(P<0.05);EMP照射引起〔Ca2+〕i荧光强度显著升高(107.34±26.14,P<0.05),MK801预处理使之有所下降(81.29±19.96,P<0.05),而MK801与尼非地平联合用药可使〔Ca2+〕i荧光强度进一步下降(69.82±25.54,P<0.05)。结论MK801和尼非地平预处理可以部分地阻断EMP所致的海马神经元损伤;细胞内钙超载在EMP损伤机制中起到重要作用。
Objective To investigate the mechanism of neuroprotective injury induced by electromagnetic pulse (EMP) in hippocampal neurons and the possibility of drug protection. Method EMP irradiation conditions of 6 × 104V · m-1, pulse rise time of 20ns, pulse width of 30μs, a frequency of 2.5 pulses · min-1, the role of time for 2min. Primary cultures of hippocampal neurons were pre-treated with MK801 and nifedipine for EMP irradiation. MTT assay was used to measure cell viability. FACS assay was used to detect apoptosis rate. Fluo-3-AM fluorescent probe loading, confocal laser scanning confocal microscope The concentration of intracellular free calcium [Ca2 +] i in hippocampal neurons was measured by scanning microscopy. Results The cell viability of MK801 group and MK801 + nifedipine group were 0.25 ± 0.06 and 0.27 ± 0.07, respectively, which were significantly higher than that of EMP group (0.17 ± 0.08, P <0.05) The fluorescence intensity of [Ca2 +] i was significantly increased (107.34 ± 26.14, P <0.05) in EMP group compared with 68.63 ± 9.04% in EMP group (P <0.05) (81.29 ± 19.96, P <0.05). The combination of MK801 and nifedipine decreased the fluorescence intensity of [Ca2 +] i (69.82 ± 25.54, P <0.05). Conclusion Pretreatment with MK801 and nifedipine can partly block EMP-induced injury of hippocampal neurons. Intracellular calcium overload plays an important role in the mechanism of EMP injury.