三七三醇皂苷对脑梗死后不同恢复时点Nogo-A表达的影响

【作者】 王席玲；

【导师】 朱陵群； 王硕仁； 邹忆怀； 高颖；

【作者基本信息】 北京中医药大学 ， 中西医结合临床基础， 2008， 硕士

【摘要】 目的通过研究三七三醇皂苷对脑梗死后不同恢复时点神经行为学及Nogo-A蛋白、Nogo-A mRNA和NgR mRNA表达的影响,并观察相应阶段突触结构特点,探讨三七三醇皂苷在脑缺血恢复期对功能重塑、重建的作用机制。方法健康雄性SD大鼠,体重300±20g。采用改良的EZ Longa方法建立大鼠大脑中动脉阻塞(MCAO)模型,待手术清醒后进行EZ Longa评分。根据EZ Longa评分随机分为模型组(MCAO手术+生理盐水)、三七三醇皂苷组(MCAO手术+三七舒通胶囊)和尼莫地平组(MCAO手术+尼莫地平)。术后麻醉清醒当天开始灌胃给药。术后5h进行神经行为学评分并做脑组织TTC染色,评价模型制做。并持续观察行为学变化至术后72h。各组于术后3d、7d和28d分别处死,迅速取左侧脑组织,通过Wester Blot方法观察Nogo-A蛋白表达变化,RT-PCR方法观察Nogo-A mRNA和NgR mRNA表达变化,透射电子显微镜观察同时间点突触结构变化。结果1.MCAO模型的制备及神经行为学评分模型组动物神经行为学评分随缺血时间延长逐渐降低,三七三醇皂苷组和尼莫地平组在各时间点均显著低于同期模型组。手术组动物神经行为学评分均在1-3分之间。入选后5 h随机取脑组织TTC染色结果示,正常组脑片红染,无白色梗死灶形成。手术组脑片有大片白色梗死灶形成,提示模型制做成功。2.三七三醇皂苷对MCAO大鼠不同恢复时点Nogo-A蛋白表达的影响脑缺血后模型组3d的Nogo-A蛋白表达较正常组增高,术后7d增加更加显著,三七三醇皂苷组和尼莫地平组在各时间点均显著低于同期模型组。3.三七三醇皂苷对MCAO大鼠不同恢复时点Nogo-A mRNA和NgR mRNA表达的影响MCAO术后模型组Nogo-A mRNA和NgR mRNA随缺血时间的延长,逐渐增高;三七三醇皂苷组和尼莫地平组缺血早期稍高,随缺血时间的延长,均显著低于同期模型组。4.三七三醇皂苷对MCAO大鼠不同恢复时点突触结构变化的影响随脑缺血时间的延长可诱导神经细胞核固缩、染色质有轻度凝聚变化,部分核膜模糊不清,线粒体明显减少、肿胀和线立体嵴断裂、突触结构发生变化。三七三醇皂苷和尼莫地平能下调轴突生长抑制因子Nogo-A的表达,起到脑保护作用,其作用在电子显微结构表现为神经细胞胞浆丰富,常染色质多;突触结构清晰,电子密度均匀;髓鞘排列紧密。结论1.三七三醇皂苷可以促进动物脑缺血后缺损神经功能的恢复(降低Longa分值)2.三七三醇皂苷可以下调脑梗死后不同恢复时点Nogo-A蛋白水平表达3.三七三醇皂苷可以下调脑梗死后不同恢复时点Nogo-A mRNA和NgR mRNA的表达4.三七三醇皂苷可以促进脑梗死后不同恢复时点突触结构的恢复即突触的重塑、重建。5.三七三醇皂苷在脑血管病不同恢复时点,促进大脑功能重塑、重建中的部分作用机制可能为:下调Nogo-A蛋白,下调Nogo-A mRNA和NgR mRNA,促进突触结构的恢复。更多还原

【Abstract】 ObjectiveIn rat MCAO (middle cerebral artery occlusion) model, we observed the effect of PTS on neuroethology and the protein level of Nogo-A, mRNA level of Nogo-A and NgR in MCAO rat brain tissue at different time point after operation. Using electron microscope, we observed eneurosynaptic structure at the same time points. Through these experiments, we aimed to explore the mechanism of injured neurons remodeling by PTS during cerebral ischemia recovery.MethodsCreate MCAO model: male SD rat (weight=300±20 g) were used in this study, apply Longa method cause middle cerebral artery occlusion. The neurological deficit grades of cerebral infarction were estimated by Longa method. After operation, rats were randomly divided to 3 groups: model group (treat with N.S), traditional Chinese medicine (TCM) group (treat with PTS) and Western medicine group (treat with nimodipine), according to Longa. N.S, PTS and nimodipine were intragastric administration once day and lasted for 3d, 7d and 28d. Nogo-A protein, Nogo-A mRNA, NgR mRNA and synaptic structure in cortex were observed by Wester Blot, RT-PCR and TEM (transmission electron microscopy) and the changes of neurological behaviour in rats with cerebral ischemia.Results:1. Production of middle cerebral artery occlusion in rats and result of grade of neuroethologyCompared with the normal group, the grade of neuroethology was significant drop at the model group with ischemic time extension. PTS group and nimodipine group in the same period were lower than model group at different time point. Scores of neurological behaviour in rats treated with MCAO were from 1 to3. TTC staining of the brain 5 h after operation demonstrated that tissue with normal blood perfusion stained red and ischemia tissue stained white, which indicated that the rat MCAO model was made successfully.2. Effect of PTS on expression of Nogo-A protein at different time after MCAO:The experimental result show that the expression of Nogo-A of model group significantly increased at 3d, and more significantly at 7d. PTS group and nimodipine group were lower than model group at the 3 different time point.3. Effect of PTS on expression of Nogo-A mRNA and NgR mRNA at different time after MCAO:The expression of Nogo-A mRNA and NgR mRNA of model group gradually increased with ischemic time extension. PTS group and nimodipine group were lower than model group at the 3 different time point,beiside of the early ischemic.4. Effect of PTS on expression of synaptic structure at different time after MCAO:With ischemic time extension,nerve cells were leaded to karyopycnosis, chromatin mild rally, part of the nuclear membrane unclear, mitochondrial obviously decreased, mitochondrial swelling and rupture ridge and synaptic structure had changed. PTS and nimodipine can be down-regulate the expression of Nogo-A, Nogo-A mRNA and NgR mRNA, which play a role in brain protection. Under the electron microscope showed cytoplasm rich, more often chromatin, synaptic structure clear, electron density and closely myelin.Conclusion:1. PTS can reduce the neurologic deficiency in rats after MCAO.2. PTS can be down-regulate the expression of Nogo-A at different time after MCAO.3. PTS can be down-regulate the expression of Nogo-A mRNA and NgR mRNA at different time after MCAO.4. PTS can enhance the synaptic remodeling at different time after MCAO.5. PTS can decrease the expression of Nogo-A、Nogo-A mRNA and NgR mRNA and enhance the synaptic remodeling at different time in rat brain after middle cerebral artery occlusion, which may be the one of remodeling mechanisms of PTS.更多还原