大鼠局灶性脑缺血后FADD和Daxx的表达  

Changes of expression of FADD and Daxx following focal cerebral ischemia in rats

作　　者：胡跃强[1] 肖波[2] 毕方方[2] 丁玲[2] 

机构地区：[1]广西中医学院第一附属医院神经内科,南宁530023 [2]中南大学湘雅医院神经内科,长沙410008

出　　处：《中华神经科杂志》2007年第11期762-767,共6页Chinese Journal of Neurology

摘　　要：目的探讨大鼠脑缺血再灌注后 Fas 相关蛋白 FADD 和 Daxx 在缺血半暗带的表达变化。方法①将 SD 大鼠随机分为假手术组和模型组。线栓法制备大鼠大脑中动脉梗阻(MCAO)模型,缺血2 h 后恢复再灌注,再灌注后1、3、6、12和24 h 处死动物。RT-PCR、免疫组织化学、Westernblot 等方法分别检测缺血半暗带 FADD、Daxx mRNA 和蛋白的表达变化;②免疫荧光双标分别标记神经元核区和 FADD 及 Daxx 蛋白染色区,激光扫描共聚焦显微镜观察二者在缺血再灌注前后神经元的定位。结果再灌注3 h 假手术组可以检测到少量的 FADD mRNA 和蛋白表达(mRNA:0.441±0.038;蛋白:156.78±13.04),模型组 FADD mRNA 和蛋白表达变化一致,在再灌注3 h 开始上升(mRNA:0.628±0.059,P<0.0l;蛋白:131.28±5.99,F=15.692,P<0.01),12 h 达到高峰(mRNA:0.971±0.070,F=56.233,P<0.01;蛋白:104.36±8.55,P<0.01),24 h 显著下降(mRNA:0.551±0.042,P<0.05;蛋白:123.01±11.87,P<0.01),模型组 FADD mRNA 和蛋白表达变化一致,在再灌注3 h 开始上升(P<0.01),12 h 达到高峰(P<0.01),24 h 显著下降(P<0.05);假手术组可以检测到较高的 Daxx mRNA 和蛋白表达;模型组 Daxx mRNA 和蛋白表达变化一致,再灌注3 h 开始上升(P<0.01),此后持续上升,一直至24 h 仍维持较高水平表达(P<0.01);激光扫描共聚焦显微镜显示脑缺血再灌注后 FADD 的免疫活性位于胞质,Daxx 的免疫活性从胞核向胞质移位。结论大鼠脑缺血急性期 FADD、Daxx mRNA 和蛋白表达增加,可能参与了脑缺血后促凋亡作用。Objective To investigate the changes of expression of Fas-associated proteins named Fas-associated death domain protein （FADD） and death-associated protein （Daxx） in the ischemic penumbra following transient focal cerebra ischemia in rats. Methods （1） Adult male Sprague-Dawley rats were randomly divided into the sham-operated group and the cerebral ischemia model group. Rats underwent right middle cerebral artery occlusion （MCAO） for 2 h and reperfusion for 1, 3, 6, 12 and 24 h using an intraluminal suture technique. The expression of FADD and Daxx mRNA and protein were measured with methods of immunohistochemistry. Western blot and reverse transcription-polymerase chain reaction （ RT-PCR） respectively were used in the ischemic penumbra of rats. （2） Double-label fluorescence confocal laser scanning microscopy （CLSM） was performed to monitor FADD and Daxx intracellular location before and after ischemia. Results RT-PCR, Immunohistochemistry, Western blot experiments indicated that a very low level of FADD mRNA and protein were detected in the cerebral cortex of sham rats. The expression level both of FADD mRNA and protein increased significantly at 3 h after reperfusion, peaked at 12 h, then declined markedly at 24 h in the ischemic penumbra of model rats. RT-PCR, Immunohistochemistry indicated that a relatively high level of Daxx mRNA was detected in the cerebral cotex of sham rats. The expression level of Daxx mRNA increased significantly at 3 h after reperfusion and persisted to 24 h at a high level, whose protein had a same change of expression level in the ischemic penumbra of model rats. Immunofluorescence double-staining laser scanning by CLSM showed that the immunoreactivity of FADD was located in cytoplasm, and the intracellular translocation of the immunoreactivity of Daxx from nucleus to cytoplasm was monitored by measuring the green fluorescence after isehemia. Conclusion The transient upregulation of FADD and the persistant high level of expression of Daxx may contribute to

关 键 词：脑缺血 再灌注损伤 衔接蛋白质类 信号传导 细胞凋亡 

分 类 号：R743.3[医药卫生—神经病学与精神病学]