高浓度氧暴露对N9小胶质细胞功能的影响  

Effect of hyperoxia exposure on the function of N9 microglia in vitro

作　　者：蒋朴[1] 徐颖[2] 胡良安[3] 刘杨[2] 邓世雄[1] 

机构地区：[1]重庆医科大学基础医学院法医学教研室,重庆400016 [2]重庆医科大学附属儿童医院麻醉科和儿童发育疾病研究省部共建教育部重点实验室,重庆400014 [3]重庆医科大学附属第一医院,重庆400016

出　　处：《南方医科大学学报》2012年第1期71-74,共4页Journal of Southern Medical University

基　　金：中国博士后基金(20090450790);重庆市自然科学基金(CSTC2007BB5293)

摘　　要：目的探讨常压高浓度氧暴露对N9小胶质细胞功能的影响。方法体外培养N9小胶质细胞,分别在900 ml/L高浓度氧中暴露2、6、12、16、24、48 h后（1）流式细胞术检测细胞存活率及凋亡率;（2）DCFH-DA荧光探针检测活性氧（ROS）含量的变化;（3）RT-PCR检测Toll样受体4（TLR4）mRNA表达变化;（4）ELISA检测N9细胞培养上清中IL-1β和TNF-α的含量;（5）免疫荧光化学染色检测TLR4蛋白的表达变化。结果流式细胞术结果示,细胞凋亡率在高氧暴露12 h后明显高于空气对照组（P〈0.05）,16 h达高峰（P〈0.01）;高氧暴露2 h后,细胞内ROS含量明显增加,呈一定时间依赖性;TLR4 mRNA及蛋白表达水平明显高于空气对照组（P〈0.05）;高氧暴露后细胞上清中TNF-α及IL-1β含量随暴露时间延长而增加,均在6 h开始升高,12~16 h达高峰（P〈0.05）,IL-1β增高较TNF-α更为明显。结论高氧暴露后N9小胶质细胞TLR4通路激活,产生大量神经毒性因子（ROS、IL-1β及TNF-α）,细胞凋亡增加。Objective To observe the effect of normobaric hyperoxia exposure on the functions of N9 microglia and explore the underlying mechanism of hyperoxia-induced immature brain injury.Methods N9 microglial cells were exposed to 900 ml/L O2 for 2,6,12,24 or 48 h,and the cell apoptotic rate was assessed using flow cytometry.The intracellular oxidative stress was measured using a fluorescent DCFH-DA probe,and the expression of Toll-like receptor 4（TLR4） mRNA was detected using RT-PCR.Interleukin-1β（IL-1β） and tumor necrosis factor-α（TNF-α） concentrations in the supernatant of the cell cultures were tested with ELISA following the exposures.TLR4 protein expression was observed using immunofluorescence staining.Results Significant cell apoptosis was detected after oxygen exposures for 12-24 h.Accumulation of reactive oxygen species（ROS） were detected after a 2-h exposure.After prolonged hyperoxia exposure,TLR4 expression and IL-1β and TNF-α levels significantly increased in the cells.Conclusion Hyperoxia exposure activates TLR4 signaling pathway in N9 microglial cells in vitro,leading to massive production of ROS,IL-1β,and TNF-α and thus triggering cell apoptosis.

关 键 词：N9小胶质细胞 常压高浓度氧 TOLL样受体4 活性氧 促炎症因子 凋亡 

分 类 号：R720.597[医药卫生—急诊医学]