【作者】 来滨；

【导师】 郑平；

【作者基本信息】 复旦大学 ， 神经生物学， 2005， 博士

【摘要】 在缺氧早期阶段,神经元发生的变化主要有线粒体呼吸链复合体抑制、细胞膜离子通道改变、神经递质释放增加、细胞内钙离子浓度增加和基因表达变化等,其中缺氧时线粒体呼吸链复合体抑制受到关注,因为近年研究发现这种抑制除了是细胞对缺氧的一种代偿反应外,它还可通过产生某些活性分子使细胞功能发生变化,从而对细胞产生保护或损伤作用。然而,目前对后一个途径还有许多不清楚的地方。本实验采用线粒体电子传递链复合体Ⅰ抑制剂rotenone、复合体Ⅱ抑制剂TTFA（TTFA, thenoyltrifluoroacetone）、复合体Ⅲ氧化中心抑制剂myxothiazol、复合体Ⅲ还原中心抑制剂antimycin A和ATP合成酶抑制剂oligomycin分别模拟缺氧时这些线粒体复合体所发生的抑制,用全细胞膜片钳技术研究这些线粒体复合体抑制后神经元持续钠电流、瞬态钠电流及细胞兴奋性的变化,并采用工具药和生化技术分析了其作用机制。结果发现:（1）线粒体电子传递链复合体Ⅰ抑制剂rotenone、复合体Ⅱ抑制剂TTFA和ATP合成酶抑制剂oligomycin对海马神经元持续钠电流无明显作用;复合体Ⅲ还原中心抑制剂antimycin A明显抑制持续钠电流,而氧化中心抑制剂myxothiazol明显增强持续钠电流;线粒体膜阴离子通道阻断剂DIDS可以阻断或翻转myxothiazol和antimvcin A的作用;抗氧化剂MPG和PHEN可以显著抑制antimycin A引起的持续钠电流减小,H₂O₂可以模拟antimycin A引起的持续钠电流减小;蛋白激酶C（PKC）抑制剂chelerythrine可以阻断myxothiazol引起的持续钠电流增大,myxothiazol可以显著提高PKC的活性。（2）复合体Ⅲ还原中心抑制剂antimycin A明显抑制瞬态钠电流,而氧化中心抑制剂myxothiazol仅略增强瞬态钠电流;抗氧化剂MPG和PHEN可以显著抑制antimycin A引起的瞬态钠电流减小、但PKC抑制剂chelerythrine对myxothiazol引起的瞬态钠电流增大无明显影响。（3）复合体Ⅲ还原中心抑制剂antimycin A明显抑制细胞的兴奋性,而氧化中心抑制剂myxothiazol则明显增强细胞的兴奋性。以上结果表明缺氧时神经元线粒体电子传递链复合体抑制对细胞功能影响非常复杂,而且它们的作用机制也可能不同,提示它们在缺氧时细胞的功能变化中可能具有重要作用。更多还原

【Abstract】 In the initial stages of hypoxia, it has been known that cells undergo alterations in mitochondrial respiratory chain, membrane ion channels, neurotransmitters release, intracellular environment and gene expression, etc. Among them, the alterations in mitochondrial respiratory chain are one focus of study. The inhibition of the complex I. complex II,complex III and complex IV of the mitochondrial respiratory chain during hypoxia has been observed in different studies. However, the downstream signaling pathways of these inhibition remain to be studied. The present paper used the complex I inhibitor rotenone. the complex II inhibitor TTFA (TTFA: thenoyltrifluoroacetone). the complex III oxidation center inhibitor myxothiazol, the complex III reduction center inhibitor antimycin A and the ATP synthase inhibitor oligomycin to mimic the inhibition of mitochondrial electron transport complex and studied the effects of these inhibition on persistent sodium currents (Inap), transient sodium currents (I_NaT) and neuronal excitability in rat hippocampal CA1 cells in slices and in acutely dissociated cells using whole cell patch-clamp methods. Moreover, we further analyzed their possible mechanisms using pharmacological approaches and biochemical technique. The results showed (1) that the complex I inhibitor rotenone, the complex II inhibitor TTFA and the ATP synthase inhibitor oligomycin had no apparent effects on I_Nap. However, the complex III oxidation center inhibitor myxothiazol significantly increased I_nap- whereas the complex III reduction center inhibitor antimycin A apparently decreased I_nap- The mitochondrial anion channel blocker DIDS alone did not have effects on I_nap. In the presence of DIDS, the increasing effect of myxothiazol on I_nap was blocked, but the decreasing effect of antimycin A was reversed. The decreasing effect of antimycin A could be cancelled by pretreatment with the antioxidant 2-mercaptopropionylglycine or 1,10 phenanthroline and H2O2 could mimic the effect of antimycin A. The increasing effect of myxothiazol was blocked by the protein kinase C inhibitor chelerythrine and protein kinase C activity is significantly increased by myxothiazol, but the antioxidant 2-mercaptopropionylglycine or 1,10 phenanthroline had no significantly influence on the effect of myxothiazol.(2) The complex III reduction center inhibitor antimycin A更多还原