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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 expression of Fas-related proteins FADD and Daxx in ischemic penumbra after cerebral ischemia-reperfusion in rats. Methods ① SD rats were randomly divided into sham operation group and model group. The middle cerebral artery occlusion (MCAO) model was established by thread occlusion. The rats were reperfused 2 h after ischemia and animals were sacrificed at 1, 3, 6, 12 and 24 h after reperfusion. RT-PCR, immunohistochemistry and Western blot were used to detect the expression of FADD and Daxx mRNA and protein in ischemic penumbra respectively; ②The immunofluorescence double labeling was used to label the neuronal nuclei and FADD and Daxx protein staining areas, Confocal microscopy was used to observe the localization of neurons before and after ischemia-reperfusion. Results A small amount of FADD mRNA and protein expression was detected at 3 h after reperfusion (mRNA: 0.441 ± 0.038; protein: 156.78 ± 13.04). The expression of FADD mRNA and protein in the model group changed at 3 h, (mRNA: 0.628 ± 0.059, P <0.01; protein: 131.28 ± 5.99, F = 15.692, P <0.01) (P <0.05). The expression of FADD mRNA and protein in the model group was similar to that of the model group (P <0.01, P <0.01) (P <0.01), peaked at 12 h (P <0.01) and decreased significantly at 24 h (P <0.05). Daxx mRNA and protein expression were higher in the sham group than in the sham group After 3h of reperfusion, the level of FADD began to increase (P <0.01), and then kept increasing until 24 h (P <0.01). The laser scanning confocal microscopy showed that the FADD immunoreactivity was located in the cell In contrast, the immunological activity of Daxx translocates from the nucleus to the cytoplasm. Conclusion The expression of FADD and Daxx mRNA and protein are increased in the acute phase of cerebral ischemia in rats, which may be involved in the pro-apoptotic effect after cerebral ischemia.