【作者】 张颖；

【导师】 李树清；

【作者基本信息】 昆明医学院 ， 神经外科学， 2006， 博士

【摘要】 目的：研究树鼩血栓性脑缺血后不同时间海马神经元损伤与线粒体应激所致细胞色素C（cytochrome C，Cyt C）表达的空间分布，同时检测海马天冬氨酸特异性半胱氨酸蛋白激酶（cysteine-containing aspartate-specific protease，caspase）mRNA含量；并观察谷氨酸（glutamate，Glu）及Ca²⁺对树鼩海马组织上述指标的影响，探讨脑缺血时海马微环境改变对线粒体应激、促凋亡蛋白释放以及caspase凋亡级联反应激活之间的相互关系，为阐明微环境异常条件下海马神经元线粒体应激的分子机制提供新的理论依据。 方法：建立光化学诱导树鼩皮层缺血模型，或单泵等速微灌流系统（single-pumped push-pull perfusingsystem，SPPPPS）进行海马微灌流，用免疫组织化学法（immunochemistry）检测缺血后不同时间缺血、灌流侧海马神经元CytC蛋白表达；同时低温差速离心分离海马脑组织线粒体和细胞质部分，用免疫印迹（western blotting）法检测Cyt C在线粒体及胞质的分布变化；实时荧光PER（real time fluorescence polymerase chain reaction）技术检测海马组织caspase-3及caspase-9 mRNA的含量。另一组动物（非缺血组）用Glu及CaCl₂微灌流海马组织，微灌流后6h于舌下静脉注射环孢霉素A（cyclosporin A，CsA）40mg·kg^-1或银杏内酯B（ginkgolide B，GB）5mg·kg^-1，观察微灌流后24h上述指标的改变。 结果：脑缺血形成后，免疫组化显示缺血4h海马神经元Cyt C蛋白表达增多，24h强烈表达，72h逐渐减少。Western blotting显示缺血侧海马组织线粒体Cyt C含量于缺血后4h开始下降（与假手术组相比，OD∶19.94降至17.48），24h时降至最低（OD∶8.24），72h略微升高（OD∶9.74）；而假手术组、缺血后4h组细胞质部分均未见Cyt C，缺血后24h胞质部分可见Cyt C（OD∶5.97）；72h时，胞质部分Cyt更多还原

【Abstract】 OBJECTIVE: To explore the hippocampal neuronal injury and the mitochondrial stress after thrombotic cerebral ischemia or glutamate and calcium solution affusing hippocampus in Tree Shrews.We observe the resulting changes in distribution of cytochrome c protein, at the same time, we detect the content of caspase （cysteine-containing aspartate-specific protease）mRNA. Our research explores how the ischemic microenvironment results in mitochondrial stress, proapoptotic protein release and activation of the caspase cascade. We provide a new molecular mechanism of mitochondrial stress in hippocampal neurons under an abnormal microenvironment.METHODS: Cerebral ischemic was induced by photochemical reaction and the right lateral hippocampus was microperfused by a kind of single-pumped push-pull perfusion system under three-dimensional orientation instrument in Tree Shrews. The expression of cytochrome c was observed in ischemic and perfused lateral hippocampi at different times by immunochemistry. Also, the hippocampus was removed, then mitochondria and cytoplasmic fragment were divided by low temperature centrifugation and the distribution of cytochrome c was assessed through western blot. We used real time fluorescence polymerase chain reaction to evaluate the relative amounts of caspase-3 and caspase-9 mRNA. In the treated group, we intravenously injected cyclosporine A （40mgkg-1） or ginkgolide B (Smg·kg^-1) at 6h after perfusing the glutamate-calcium chloride solutions into the hippocampus and inspected the above-mentioned items.RESULTS: Cytochrome c immunoreactivity was present in the cytosol of neurons at 4h after thrombosis, increased at 24h and decreased at 72h. Compared with sham group （Just iv rose bengal, not illumination）, Western blot results demonstrated mitochondrial levels of Cyto C lessened at 4h after thrombosis (optical density,OD:更多还原