【作者】 梁辉；

【导师】 蔡定芳；

【作者基本信息】 复旦大学 ， 中西医结合临床， 2003， 博士

【摘要】 大量动物实验表明，急性脑缺血状态下，神经元谷氨酸转运体（EAAC1）发生逆向转运，导致细胞外谷氨酸升高。谷氨酸激活突触后兴奋性氨基酸受体介导兴奋性氨基酸毒性，级联反应以此为始触发钙超载、炎症、程序性细胞死亡等下游一系列反应事件的发生,导致神经元死亡。虽然各种有效降低兴奋毒性的方法，均能有效保护神经元，但兴奋性氨基酸受体拮抗剂由于有严重毒副作用，临床使用受到限制。中西医结合治疗急性缺血性中风具有广泛前景。早期选用芳香开窍方案治疗，已证实有较好的疗效。本研究从EAAC1角度，探讨了芳香开窍药物麝香的主要成份—麝香酮对急性脑缺血损伤的神经保护作用及可能作用机理。目的 论证EAAC1对急性脑缺血损伤大鼠脑损伤的促进作用，探讨麝香酮对急性脑缺血损伤大鼠的神经保护作用并阐明其可能机理。方法 采用脑内微量注射技术向大鼠注射EAAC1反义寡核苷酸（Antisense），建立大脑中动脉栓塞（MCAO）模型，运用Western blot法和TTC染色观察缺血区EAAC1表达和梗塞体积，探讨EAAC1对急性脑缺血损伤大鼠的影响；在此基础上，运用TTC染色和透射电镜，观察麝香酮对MCAO大鼠梗塞体积和皮层超微结构的作用；为探讨麝香酮作用机理，运用RT-PCR法和Western blot法观察麝香酮对MCAO大鼠海马和大脑皮层EAAC1基因表达、蛋白表达的影响；同时，通过DNA重组技术建立了反映EAAC1功能的细胞模型，观察不同浓度麝香酮对EAAC1逆转运活性的影响。结果 反义寡核苷酸(Antisense)组EAAC1表达低，梗塞体积为105.67±8.7mm3，而正义寡核苷酸（Sense）组和生理盐水（NS）组EAAC1表达高，梗塞体积分别为180.83±15.7 mm3和182.67±13.7 mm3。Antisense组梗塞体积明显小于各对照组（P<0.01）。提示EAAC1可促进急性缺血性中风脑损伤。 MCAO模型组脑梗塞体积为182.7±18.5mm3，麝香酮组脑梗塞体积为131.2±19.6mm3,差别有显著性意义（P<0.01）。电镜显示 MCAO组核染色质丢失，胞质溶解破坏。麝香酮组见线粒体，内质网扩张，线粒体肿涨，部分有嵴丢失，核染色质正常,较模型组病变明显为轻。提示麝香酮对MCAO大鼠具有神经保护作用。<WP=5> 与假手术组相比，MCAO大鼠海马EAAC1mRNA表达在缺血6小时、24小时持续增高（p<0.01）。大脑皮层EAAC1mRNA表达在缺血24小时表达升高（p<0.01）。在大鼠海马和大脑皮层，EAAC1蛋白在缺血24小时表达升高(p<0.01)。麝香酮可下调海马和大脑皮层EAAC1mRNA表达和蛋白表达的升高（p<0.05或p<0.01）。与EAAC1逆转运模型组相比，麝香酮浓度为0.01%和0.02%时，对EAAC1逆转运活性无影响（p>0.05），浓度为0.04%和0.08%时可抑制EAAC1逆转运活性（p<0.01），至浓度为0.16%时抑制作用开始下降（p<0.01）。提示麝香酮可下调EAAC1表达和抑制EAAC1逆向转运。结论：(1) EAAC1可促进急性缺血性中风脑损伤。（2）麝香酮可减少MCAO大鼠梗塞体积，改善缺血皮层超微结构变化。（3）麝香酮的作用可能与下调EAAC1表达和抑制EAAC1逆向转运有关。更多还原

【Abstract】 Neuronal death after cerebral is determined by damage cascade including exitotoxicity, calcium over- loading, oxygen free radical expression, inflammation and so on. During cerebral ischemia, there is a rise in extracelluar cerebral glutamate content that activate glutamate- mediated channels to neurotoxic levels. Recent studies demonstrate that reversal of neural excitatory amino acid transporter(EAAC1) instead of uptake to be a principal cause for elevated extracellular glutamate levels that induce excitotoxity and inniate damage cascade. Therefore, anti-excitotoxity possesses the action of neuro -protection. Xingnaokaiqiao formula was wildly used in the early stage of cerebral ischemia. In this study , we observed whether Muscone could attenuate EAAC1 expression and reverse uptake during cerebral ischemia and participate in neuprotection .Further, the research would suggest a new clew for anti-excitotoxity. Methods Brain microinjection of neural excitatory amino acid transporter(EAAC1) antisense oligodeoxyneucleotide（EAAC1 antisense）, TTC staining and western blot analysis were adopted for probing the effects of EAAC1 on the brain injury in rat model with MCAO. In order to study its neuroprotection,Muscone was given(1mg/kg b.w) by gavage before middle cerebral artery occlusion(MCAO) operation. At 24h after MCAO, brain tissues were taken by decapitation and infarction volumes were qualified by TTC staining. The change of ultrastructure with cerebral cortex were observed by electron microscope.Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of different group. In order to study Muscone’s neuroprotection mechanism, Semi-quantitative analysis of RT-PCR and western blot analysis were used to detect EAAC1 mRNA and protein expression of EAAC1、GLT of<WP=7>different group. At the same time,the effect of muscone (0.01% to 0.16%) on reversed uptake glutamate was examined.Results: After microinjection of EAAC1 antisense into the central area of cerebral ischemia , the expression of EAAC1 was nearly depressed （P<0.01）and the volume of brain ischemic infarct was reduced（P<0.01）.Treatment with Muscone significantly reduced the infarction volume and ameliorate the damage of ultrastructure. RT-PCR and western blot analysis showed that EAAC1 mRNA expression began to increase gradually at 6hr（p<0.01） while protein expression was higher at 24hr only in hippocampus after MCAO（p<0.01） .Both EAAC1 mRNA in cerebral cortex and protein expression in hippocampus and cerebral were higher at 24hr only after MCAO（p<0.01）. In the Muscone-treated rats , a marked reduction was shown in higher levels of EAAC1 mRNA levels and protein levels at different time after MCAO（p<0.05or p<0.01）.Muscone at concentration 0.04% and 0.08%began to attenuate EAAC1 reverse uptake（p<0.01）while at concentration 0.16%, its action began to decreased（p<0.05）. Conclution our data indicated that EAAC1 is related to brain damage during cerebral ischemia. Muscone could attenuate infarct volume and ameliorate the damage of ultrastructure with cerebral cortex, so it possesses the action of neuprotection. Moreover, the neuroprotection is possibly related to attenuate EAAC1 expression and reverse uptake during cerebral ischemia更多还原