目的 研究L-型钙通道拮抗剂双苯氟嗪在大鼠全脑缺血再灌注后海马CA1区神经元中的抗凋亡作用及与CD95(Fas)分子启动的死亡信号转导通路之间的关系。方法 四血管阻断法制备全脑缺血再灌注损伤模型(缺血15 min，再灌注3 d)，双苯氟嗪（20，40和80 mg·kg-1）每日灌胃给药1次。激光扫描共聚焦显微镜(Confocal)和HE染色观察海马CA1区神经元细胞形态学改变，流式细胞仪(FCM)分析海马DNA含量变化；Western-blotting和RT-PCR方法检测CD95(Fas)分子启动的死亡信号转导通路相关蛋白Fas,FasL和Caspase3蛋白及mRNA表达变化。结果 全脑缺血再灌注后海马CA1区神经元呈现典型凋亡细胞形态学改变，凋亡细胞百分率明显上升（P<0.01）。双苯氟嗪明显改善海马CA1区神经元形态，显著降低凋亡细胞百分率（P<0.01）。Western blotting及RT-PCR结果显示，双苯氟嗪显著降低CD95(Fas)分子相关蛋白Fas、FasL和Caspase3在蛋白及核酸水平的表达。结论 双苯氟嗪对缺血性脑损伤的保护作用可能通过抑制CD95(Fas)分子启动的死亡信号转导通路，从而发挥其抗脑缺血再灌注损伤作用。
Abstract
OBJECTIVE To investigate whether dipfluzine (Dip) plays an anti-apoptotic role in the hippocampal CA1 region after transient forebrain ischemia and the relationship between the anti-apoptotic role of DIP and the death signaling transduction pathway initiated by CD95 （Fas） molecules METHODS Transient forebrain ischemia model in rats （15 min ischemia followed by 3 days reperfusion） was set up using the four-vessel method. The animals were divided randomly into five groups: shame control group, ischemia/reperfusion (I/R) group, DIP treatment group (20, 40, 80 mg·kg-1 body weight ig, respectively). The effect of DIP on neuronal apoptosis in the rat hippocampal CA1 region was studied by light microscopy, confocal and flow cytometry analysis Western blotting and RT-PCR were performed for detecting the death signaling transduction pathway initiated by CD95 （Fas） molecules, such as the expression changes of Fas, FasL and Caspase3 molecules in protein and mRNA levels RESULTS Neurons in the rat hippocampal CA1 region after transient forebrain ischemia showed typical apoptotic features under light microscopy and confocal. The morphology was improved greatly after the treatment of DIP; Sub-G1 peak was found by flow cytometry analysis and the apoptosis index decreased after the treatment of DIP (P<0.01). The expression of Fas, FasL and Caspase3 in protein and mRNA levels increased after ischemia and reperfusion (P<0.01), and decreased after the treatment of 20 mg·kg-1 or 40 mg·kg-1 of DIP In DIP (80 mg·kg-1) group, the expression of Fas, FasL protein showed no significant differences compared with I/R group （P>0.05） CONCLUSION DIP tends to have anti-apoptotic role in the rat hippocampal CA1 region subjected to transient forebrain ischemia through inhibiting the death signalling transduction pathway initiated by CD95 molecules which was closely associated with Fas, FasL and Caspase3 in protein and mRNA levels.
关键词
双苯氟嗪 /
CD95 /
死亡信号转导通路
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Key words
dipfhuzine /
CD95 /
death signaling transduction pathway
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参考文献
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脚注
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基金
国家863高技术研究发展计划资助项目(2002AA2Z3132)
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