MK801抑制乐果诱导的大鼠皮层神经元凋亡的作用研究  

Antagonism effect of MK801 on apoptosis of primary-cultured cortical neuron caused by dimethoate

作　　者：崔红梅[1] 常秀丽[1] 徐甫[1] 吴庆[1] 周志俊[1] 

机构地区：[1]复旦大学公共卫生学院教育部公共卫生安全重点实验室,上海200032

出　　处：《卫生研究》2011年第5期568-572,共5页Journal of Hygiene Research

基　　金：国家科技支撑计划(No.2006BAI06B01);上海市公共卫生重点学科建设项目(No.08GWZX0303);上海市学科带头人培养计划(No.08GWD20)

摘　　要：目的通过应用N-甲基-D-天门冬氨酸(NMDA)受体非竞争性拮抗剂MK801探讨兴奋性氨基酸神经递质在乐果诱导的新生大鼠皮层神经元凋亡中发挥的作用。方法在纯化的新生鼠皮层神经元中,加入终浓度为100μmol/L的乐果,并用50和100μmol/L NMDA受体非竞争性拮抗剂MK801对100μmol/L乐果染毒组进行干预。染毒后48小时收获细胞,TUNEL染色检测神经元凋亡情况;HPLC-FLD方法测定细胞内兴奋性氨基酸递质含量,RT-PCR检测NMDA受体NR2B亚基mRNA表达的变化,荧光探针DCFH-DA试剂盒检测细胞内活性氧水平。结果在纯化培养的皮层神经元中,100μmol/L乐果染毒48h后,与对照组相比,Tunel染色强度为对照组1.40倍(P<0.01);兴奋性氨基酸的含量均上升(P<0.01);细胞内ROS水平逐渐增高为对照组的2.47倍(P<0.01)。对100μmol/L乐果染毒组给予50和100μmol/L MK801干预后,高剂量干预组凋亡减少为干预前的79.6%(P<0.01),EAA含量下降(P<0.01);细胞内ROS水平下降为干预前的88.9%和74.8%(P<0.01),但仍远高于对照组细胞内活性氧水平(P<0.01);NR2B mRNA表达上升为干预前的1.59和2.22倍(P<0.01),显著高于对照组水平(P<0.01)。结论兴奋性氨基酸递质和细胞内活性氧共同参与乐果诱导的神经元凋亡。NMDA受体阻断剂MK801不仅能减少活性氧的生成并能降低神经元兴奋性氨基酸含量,从而减少乐果诱导的神经元凋亡。Objective To explore the antagonism effect of MK801 on apoptosis of primary-cultured cortical neurons caused by dimethoate.Methods Cortical neurons were isolated and cultured in serum free medium for 6 days in vitro,then treated with 100μmol/L dimethoate for 48h.50 and 100μmol/L MK801,a N-methyl-D-aspartic acid receptor（NMDAR）blocker,were used to intervene 100μmol/L dimethoate group.HPLC-FLD was taken to measure the concentration of excitatory amino acid.RT-PCR was used to detect transcription level of NR2B.Florescence probe DCFH-DA kit was used to detect ROS level.TUNEL staining was used to determine apoptosis.Results When neurons were treated with 50 and 100μmol/L MK801 to 100μmol/L dimethoate group,Apoptosis decreased to 79.6% of 100μmol/L dimethoate group in higher intervention group（P0.01）.Concentration of EAA decreased in both intervention groups（P0.01）.The level of ROS decreased to 88.9% and 74.8% of 100μmol/L dimethoate group（P0.01）,while still higher than that of control group（P0.01）.Transcription level of NR2B increased to 1.59 and 2.22 folds of 100μmol/L dimethoate group（P0.01）and had big differences with control group（P0.01）.Conclusion Excitatory amino acid system and ROS both contribute to the apoptosis of neurons caused by dimethoate.MK801 not only decrease the ROS level but also diminish the elevated excitatory amino acid concentration caused by dimethoate,thus abolish apoptosis of neuron.

关 键 词：乐果 凋亡 N-甲基-D-天门冬氨酸受体 MK801 皮层神经元 

分 类 号：Q593.2[生物学—生物化学] R139.3[医药卫生—劳动卫生]