摘要：
目的 研究电磁脉冲( electromagnetic pulse,EMP)对小鼠BV-2小胶质细胞形态及分泌功能的影响,并初步探讨其作用机制.方法 离体培养的BV-2细胞经200 kV/m EMP辐照200次,分别在辐照后1、6、12、24h收集细胞培养上清及细胞.倒置显微镜下观察细胞形态变化,ELISA法检测培养上清中肿瘤坏死因子-α(TN F-α)、白细胞介素(IL)-1β、IL-10等细胞因子水平的变化,硝酸还原酶法检测培养上清中一氧化氮(NO)水平,DCFH-DA探针检测活性氧,免疫印迹(Western-blot)法检测细胞外信号调节激酶(ERK)、c -Jun氨基末端激酶(JNK)、p38磷酸化水平和蛋白表达量的变化.应用p38抑制剂( SB203580)预处理细胞后再进行EMP辐照,然后检测培养上清中NO水平和活性氧的生成.结果 EMP辐照后1、6和12h,部分小胶质细胞出现胞体变大、突触变粗变短,且活化细胞比例与假辐照组相比明显增加,差异有统计学意义(P＜0.05)；EMP辐照后细胞培养上清中TNF-α、IL-1β、IL-10等细胞因子水平未发生明显改变,但活性氧检测结果显示,与假辐照组(小胶质细胞平均荧光强度10.34)相比,EMP辐照后1h小胶质细胞荧光强度(平均荧光强度21.56)明显增加,6h达峰值(平均值为32.46),12h开始恢复(平均荧光强度24.36),差异均有统计学意义(P＜0.05),24h恢复至假辐照水平；EMP辐照后NO水平的变化与活性氧一致,辐照后1h开始增加,6h达峰值,12h开始恢复,24h恢复至假辐照组水平；蛋白杂交结果显示,EMP辐照后1、6h,p38的磷酸化水平和蛋白水平较假辐照组明显增加,差异有统计学意义(P＜0.05),ERK和JNK无明显变化.应用p38抑制剂SB203580预处理细胞,明显抑制了EMP诱导的小胶质细胞对活性氧和NO的产生,活性氧水平除6h组未恢复至假辐照水平外,其他各组均恢复至假辐照水平,NO水平各组均恢复至假辐照组水平.结论 EMP辐照可活化小胶质细胞并且促进其对NO和活性氧的生成,p38信号通路参与了此过程.%Objective To study the effects of electromagnetic pulse (EMP) exposure on the morphological change and excretion functions of mouse microglia (BV-2) cells and possible mechanism.Methods BV-2 cells were divided into two groups:the group exposed to EMP at 200 kV/m for 200 pulses and sham exposure group.At 1,6,12 and 24 hour after exposure the cells and culture supernatant were collected.Cellular morphological change was observed under invert microscope,the levels of TNF-α,IL-1β and IL-10 in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA),nitric oxide (NO) and reactive oxygen species (ROS) were detected by nitrate reductase method and DCFH-DA probe,respectively.The protein and phosphorylation levels of ERK,JNK and p38 were measured by Western Blot method.After the cells pretreated with the inhibitor of p38 (SB203580) were exposed to EMP,the levels of NO and ROS in culture supernatant were detected.Results It was found that the large ameboid shape appeared in some microglia cells exposed to EMP for 1,6 and 12 h.Moreover,the number of microglia cells with ameboid shape increased significantly at 1 h,6 h and 12 h after EMP exposure compared with sham group (P＜0.05).The levels of cytokines,such as TNF-α,IL-1β and IL-10,in culture supernatant did not change obviously after EMP exposure.The levels of NO and ROS increased significantly at 1 h after EMP exposure,reached the peak at 6 h,began to recover at 12 h and recovered to sham group level at 24 h (P＜0.05).Western blot results showed that the protein and protein phosphorylation levels of ERK and JNK did not change significantly after EMP exposure,however,the protein and protein phosphorylation levels of p38 increased obviously at 1 h and 6 h after EMP exposure,compared with sham group (P＜0.05).In addition,the pretreatment of p38 inhibitor (SB203580) significantly decreased NO and ROS production induced by EMP.Conclusion EMP exposure may activate microglia cells and promote the production of NO and ROS in mouse microglia cells,and p38 pathway is involved in this process.
