微波辐照对血管内皮细胞转录因子NF-E2相关因子2磷酸化及蛋白激酶C活性的影响(英文)

Microwave radiation affects the phosphorylation ofnuclear factor erythroid-2-related factor-2 and activity of protein kinase C in vascular endothelial cells

背景:转录因子NF-E2相关因子2是细胞调节抗氧化应激反应的重要转录因子,有实验表明转录因子NF-E2相关因子2能被蛋白激酶C家族中的众多成员磷酸化,为进一步研究微波辐射造成氧化应激损伤的产生机制,是否可以从转录因子NF-E2相关因子2途径观察微波辐照对抗氧化调控系统的影响?目的:分析微波辐照对血管内皮细胞转录因子NF-E2相关因子2的磷酸化及蛋白激酶C活性的影响。设计:观察对比实验。单位:解放军第三军医大学劳动卫生学教研室。材料:血管内皮细胞株,H332PO4,Protein-A Sepharose(Sigma公司),转录因子NF-E2相关因子2单抗(H-300,Santa Cruz),蛋白激酶Cα单抗(Santa Cruz),玻纤滤膜(Whatman公司)凝胶扫描系统(Gel Doc 2000,Bio-Rad),液闪测定仪(LKB-117瑞典)。方法:实验于2003-03/07在解放军第三军医大学劳动卫生学教研室电磁辐射生物效应研究室完成。①转录因子NF-E2相关因子2磷酸化分析:血管内皮细胞以DMEM培养液培养至细胞生长至旺盛期,加入32Pi孵育标记2h,将细胞培养瓶置于37℃水浴盒中,在反射系数近似零的微波暗室接受微波辐照,为辐照组,辐照平均功率密度为30mW/cm2,辐照时间30min;以未接受辐照的细胞为对照组。采用免疫共沉淀-放射自显影法测定转录因子NF-E2相关因子2磷酸化水平,用凝胶扫描系统对细胞转录因子NF-E2相关因子2磷酸化水平进行半定量分析,对照组细胞直接进行分析。②蛋白激酶C蛋白激酶活性分析及表达量测定:辐照组及对照组细胞培养方法、条件,辐照方式、剂量、条件同前。于辐照后2,4,8,24h裂解细胞,提取胞浆和胞膜蛋白,采用r-32P-ATP标记液闪法进行蛋白激酶C活性测定;采用凝胶扫描系统对蛋白条带灰度进行半定量分析;对细胞进行蛋白激酶C免疫细胞化学染色后于光镜下观察细胞胞浆的着色程度,对照组均直接给予测定和观察。主要观察指标:①辐照组及对照组转录因子NF-E2相关因子2磷酸化水平检测结果。②细胞蛋白激酶活性分析及表达量测定结果。结果:①辐照组及对照组转录因子NF-E2相关因子2磷酸化水平测定结果:辐照组辐照后2,4,8h,转录因子NF-E2相关因子2条带灰度强于对照组细胞,辐照4h,转录因子NF-E2相关因子2磷酸化达到峰值水平,条带灰度扫描半定量分析显示辐照2,4,8h辐照组细胞转录因子NF-E2相关因子2磷酸化水平较对照组分别增加33%,261%,141%(t=2.974,4.209,4.047,P<0.05),24h恢复正常。②辐照组及对照组细胞蛋白激酶C表达量及活性检测结果:微波辐照2,4,8h,辐照组细胞蛋白激酶C表达量高于对照组,以4h最明显,辐照后24h恢复正常;免疫细胞化学染色显示辐照组细胞辐照4h胞浆着色强度强于对照组;r-32P-ATP标记液闪法显示辐照2,4,8h,辐照组细胞蛋白激酶C活性较对照组分别增加了36%,93%,47%(t=2.801,3.654,3.035,P<0.05),24h有回落。蛋白激酶C激活的峰值发生在照射后4h。结论:微波辐照可引起血管内皮细胞转录因子NF-E2相关因子2磷酸化水平在一定时段内增强,同时可引起细胞蛋白激酶C表达增加,其活性变化时间效应与转录因子NF-E2相关因子2磷酸化水平有一致性。

BACKGROUND： Nuclear factor erythroid-2-related factor-2 （NF-E2-related factor-2） is an important transcription factor to regulate anti-oxidative stress reaction. Some researches indicate that NF-E2-related factor-2 can be phosphorylated by numerous members of protein kinase C family. In order to investigate generant mechanism of microwave radiation on oxidative stress injury, whether microwave radiation can influence on anti-oxidative regulating system through NF-E2-related factor-2 or not should be further studied. OBJECTIVE： To analyze the effect of microwave radiation on phosphorylation of NF-E2-related factor-2 and activity of protein kinase C in vascular endothelial cells. DESIGN： Observational-contrast study. SETTING： Department of Labor Hygiene, the Third Military Medical University of Chinese PLA. MATERIALS： Vascular endothelial cell strain; H332PO4; Protein-A Sepharose （Sigma Company）; mono-antibody of NF-E2-related factor-2 （H-300, Santa Cruz）; α-mono-antibody of protein kinase C （Santa Cruz）; glass microfiber filters （Whatman Company）; gel scanning system （Gel Doc 2000, Bio-Rad）; liquid scintillation spectrometer （LKB-117, Sweden）. METHODS： The experiment was carried out in Laboratory of Electromagnetic radiation and Biological Effect, Department of Labor Hygiene, the Third Military Medical University of Chinese PLA from March to July 2003. ① Analysis of phosphorylation of NF-E2-related factor-2： Vascular endothelial cells were cultured with DMEM medium till the period of productive growth and incubated with ^32Pi for 2 hours. And then, cultured bottle was maintained in water bath at 37% and performed with microwave radiation in dark chamber, whose reflectivity was about zero. It was regarded as radiation / group, and the average power density of radiation was 30 mW/cm^2; in addition, the duration of radiation was 30 minutes. Cells did not deal with microwave radiation were regarded as control group. Phosphorylation level of NF-E2-related factor-2 was measured at 2, 4, 8 and 24 hours after radiation with immune coprecipitation-autoradiography technique and dealt with semi-quantitative analysis with gel scanning system. Cells in the control group were analyzed directly. ② Active analysis and expressional measurement of protein kinase C： Cells in the radiation group and the control group were dealt with the same cultured method, condition, radiation styles, dosage and environment as mentioned above. At 2, 4, 8 and 24 hours after radiation, cells were split to extract plasma and membrane protein. Furthermore, activity of protein kinase C was measured with r-^32P-ATP labeled liquid scintillation spectrometer; gray value of protein strap was dealt with semi-quantitative analysis with gel scanning system; staining degree of plasma was observed after immunocytochemical staining of protein kinase C. In addition, cells in the control group were measured and observed directly. MAIN OUTCOME MEASURES ： ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group; ② Results of active analysis and expressional measurement of protein kinase C in radiation group and control group. RESULTS： ① Phosphorylation level of NF-E2-related factor-2 in radiation group and control group： Gray value of NF-E2-related factor-2 was higher in radiation group than that in control group at 2, 4 and 8 hours after radiation. Phosphorylation level of NF-E2-related factor-2 reached the peak at four hours after radiation. In addition, results of semi-quantitative scanning analysis showed that, at 2, 4 and 8 hours after radiation, phosphorylation level of NF-E2-related factor-2 was increased 33%, 261% and 141% in radiation group as compared with that in control group, respectively （t = 2.974, 4.209, 4.047, P 〈 0.05）, and then, fallen down to normal value 24 hours later. ② Results of active analysis and expressional measurement of protein kinase C in radiation group and control group： At 2, 4 and 8 hours after microwave radiation, expression of protein kinase C in radiation group was higher than that in control group, especially at the 4 hour. In addition, at 24 hours after radiation, expression of protein kinase C recovered the normal value. Results of immunocytochemical staining showed that staining of plasma was deeper in radiation group than that in control group at 4 hours after radiation： Moreover, results of r-^32P-ATP labeled liquid scintillation spectrometer also suggested that, at 2, 4 and 8 hours after radiation, activity of protein kinase C was increased 36%, 93% and 47% in radiation group as compared with that in control group, respectively （t =2.801, 3.654, 3.035, P 〈 0.05）. And then, activity of protein kinase C was decreased after 24 hours, otherwise, activity of protein kinase C reached the peak at 4 hours after radiation. CONCLUSION： Microwave radiation can strengthen the phosphorylation level of NF-E2-related factor-2 in vascular endothelial cells during a special period; meanwhile, it can also cause the increase of expression of protein kinase C. Time effect of activity of protein kinase C is coincidence with phosphorylation level of NF-E2-related factor-2.