中华劳动卫生职业病杂志
中華勞動衛生職業病雜誌
중화노동위생직업병잡지
CHINESE JOURNAL OF INDUSTRIAL HYGIENE AND OCCUPATIONAL DISEASES
2012年
3期
163-167
,共5页
杨龙龙%周艳%李海娟%郭娟%张琰君%丁桂荣%郭国祯
楊龍龍%週豔%李海娟%郭娟%張琰君%丁桂榮%郭國禎
양룡룡%주염%리해연%곽연%장염군%정계영%곽국정
电磁场%神经胶质细胞%活性氧组分%一氧化氮%有丝分裂素激活蛋白激酶类
電磁場%神經膠質細胞%活性氧組分%一氧化氮%有絲分裂素激活蛋白激酶類
전자장%신경효질세포%활성양조분%일양화담%유사분렬소격활단백격매류
Electromagnetic fields%Microglia%Reactive oxygen species%Nitric oxide%Mitogen-activated protein kinases
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.
目的 研究電磁脈遲( electromagnetic pulse,EMP)對小鼠BV-2小膠質細胞形態及分泌功能的影響,併初步探討其作用機製.方法 離體培養的BV-2細胞經200 kV/m EMP輻照200次,分彆在輻照後1、6、12、24h收集細胞培養上清及細胞.倒置顯微鏡下觀察細胞形態變化,ELISA法檢測培養上清中腫瘤壞死因子-α(TN F-α)、白細胞介素(IL)-1β、IL-10等細胞因子水平的變化,硝痠還原酶法檢測培養上清中一氧化氮(NO)水平,DCFH-DA探針檢測活性氧,免疫印跡(Western-blot)法檢測細胞外信號調節激酶(ERK)、c -Jun氨基末耑激酶(JNK)、p38燐痠化水平和蛋白錶達量的變化.應用p38抑製劑( SB203580)預處理細胞後再進行EMP輻照,然後檢測培養上清中NO水平和活性氧的生成.結果 EMP輻照後1、6和12h,部分小膠質細胞齣現胞體變大、突觸變粗變短,且活化細胞比例與假輻照組相比明顯增加,差異有統計學意義(P＜0.05)；EMP輻照後細胞培養上清中TNF-α、IL-1β、IL-10等細胞因子水平未髮生明顯改變,但活性氧檢測結果顯示,與假輻照組(小膠質細胞平均熒光彊度10.34)相比,EMP輻照後1h小膠質細胞熒光彊度(平均熒光彊度21.56)明顯增加,6h達峰值(平均值為32.46),12h開始恢複(平均熒光彊度24.36),差異均有統計學意義(P＜0.05),24h恢複至假輻照水平；EMP輻照後NO水平的變化與活性氧一緻,輻照後1h開始增加,6h達峰值,12h開始恢複,24h恢複至假輻照組水平；蛋白雜交結果顯示,EMP輻照後1、6h,p38的燐痠化水平和蛋白水平較假輻照組明顯增加,差異有統計學意義(P＜0.05),ERK和JNK無明顯變化.應用p38抑製劑SB203580預處理細胞,明顯抑製瞭EMP誘導的小膠質細胞對活性氧和NO的產生,活性氧水平除6h組未恢複至假輻照水平外,其他各組均恢複至假輻照水平,NO水平各組均恢複至假輻照組水平.結論 EMP輻照可活化小膠質細胞併且促進其對NO和活性氧的生成,p38信號通路參與瞭此過程.
목적 연구전자맥충( electromagnetic pulse,EMP)대소서BV-2소효질세포형태급분비공능적영향,병초보탐토기작용궤제.방법 리체배양적BV-2세포경200 kV/m EMP복조200차,분별재복조후1、6、12、24h수집세포배양상청급세포.도치현미경하관찰세포형태변화,ELISA법검측배양상청중종류배사인자-α(TN F-α)、백세포개소(IL)-1β、IL-10등세포인자수평적변화,초산환원매법검측배양상청중일양화담(NO)수평,DCFH-DA탐침검측활성양,면역인적(Western-blot)법검측세포외신호조절격매(ERK)、c -Jun안기말단격매(JNK)、p38린산화수평화단백표체량적변화.응용p38억제제( SB203580)예처리세포후재진행EMP복조,연후검측배양상청중NO수평화활성양적생성.결과 EMP복조후1、6화12h,부분소효질세포출현포체변대、돌촉변조변단,차활화세포비례여가복조조상비명현증가,차이유통계학의의(P＜0.05)；EMP복조후세포배양상청중TNF-α、IL-1β、IL-10등세포인자수평미발생명현개변,단활성양검측결과현시,여가복조조(소효질세포평균형광강도10.34)상비,EMP복조후1h소효질세포형광강도(평균형광강도21.56)명현증가,6h체봉치(평균치위32.46),12h개시회복(평균형광강도24.36),차이균유통계학의의(P＜0.05),24h회복지가복조수평；EMP복조후NO수평적변화여활성양일치,복조후1h개시증가,6h체봉치,12h개시회복,24h회복지가복조조수평；단백잡교결과현시,EMP복조후1、6h,p38적린산화수평화단백수평교가복조조명현증가,차이유통계학의의(P＜0.05),ERK화JNK무명현변화.응용p38억제제SB203580예처리세포,명현억제료EMP유도적소효질세포대활성양화NO적산생,활성양수평제6h조미회복지가복조수평외,기타각조균회복지가복조수평,NO수평각조균회복지가복조조수평.결론 EMP복조가활화소효질세포병차촉진기대NO화활성양적생성,p38신호통로삼여료차과정.
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.
