【作者】 周贞洁；

【导师】 夏若虹；

【作者基本信息】 华东师范大学 ， 生物物理学， 2006， 硕士

【摘要】 在人类健康问题被异常关注的今天，氧化还原环境对生命进程和质量的影响正成为相关领域研究的热点。研究发现，各类理化因子对生物体内的自由基水平产生了深远的影响，然而其内在的微观影响机制，例如胞内氧化还原机制与钙调控机制之间的关系仍有待深入的探讨。加之，由于细胞水平的研究存在重复性差、模型复杂的特点，分子、细胞器水平的相关研究工作就显得尤为必须和重要。 本文以提取的兔骨骼肌肌质网（SR）囊泡为研究对象，探讨两种特定的理化因子（50Hz 04mT磁场和Na⁺、K⁺生理盐离子）对SR囊泡自由基产生机制的调控作用及其对SR上钙释放通道（ryanodine receptor，RyR1）的影响，并初步考察氧化还原系统在SR囊泡上的具体活性位点。 实验采用荧光分光光度计、紫外—可见分光光度计检测两种理化因子对SR上NADH氧化酶（NOX）活性以及伴生超氧自由基（O₂^-）产率的影响；采用[³H]-ryanodine结合实验、钙释放动力学实验检测两种理化因子对SR上RyR1功能状态的影响；利用去垢剂的作用确定NOX在SR上的位置。 结果显示，高浓度的Na⁺、K⁺对SR上NOX活性（由NADH的氧化初速率表征）以及伴生的O₂^-产率均有抑制作用；在[³H]-ryanodine结合实验中，高浓度的Na⁺、K⁺也抑制了由NADH诱导的ryanodine与RyR1结合的升高趋势；在钙释放动力学实验中，不同浓度的K⁺调控了由NADH诱导的Ca²⁺释放初速率变化：NOX活性以及O₂^-产率与RyR开放程度存在正相关关系。同样，50Hz 0.4mT磁场辐照30min，有效地促进了NADH的氧化以及O₂^-的产生，由NADH诱导的受配体的结合能力以及RyR1的Ca²⁺释放速率也在磁场作用下得以相应提高。而去垢剂作用使外源的NADH迅速进入SR囊泡膜内，不与膜外的NOX产生氧化还原反应。 由此猜测，骨骼肌肌质网囊泡上存在具有对特定理化因子敏感并中介超氧自由基产生的NADH氧化还原系统，其参与调节肌质网钙释放通道的活性，并且这一氧化还原系统的关键酶——NOX可能在SR囊泡膜上靠外侧的一面。更多还原

【Abstract】 The isolated SR vesicles were used to determine whether the effects of NADH on activation of the sarcoplasmic reticulum ryanodine receptor （RyR1） were modulated by the Specific Physical and Chemical Factors: salt ions （Na⁺ and K⁺） and 50Hz 0.4mT magnetic field （MF）.In this research, fluorescence spectrophotometer and UV spectrophotometer were used to measure the initial rates of NADH oxidation and the production of superoxide ions in different physical and chemical conditions; the [³H]-ryanodine binding rate, the Ca²⁺ release rate were also detected in the different physical and chemical conditions, which showed the effects of the two facts on RyR1.The results showed that the initial rates of NADH oxidation and the production of superoxide were depressed at higher concentrations of Na⁺ and K⁺, exerted a salt ions concentration dependent manners; the [³H]-ryanodine binding assays showed that the activations of RyR1 induced by NADH, were depressed at higher salt concentrations; the Ca²⁺ release initial rates were also modulated by the different concentrations of KCl; all the results indicated that the function of RyR1 were correlative with the NOX activity and the superoxide production. The effects of 50Hz 0.4mT MF were similar to those of the salt ions. The initial rates of NADH oxidation and superoxide production were obviously increased by the 30 minutes MF exposure. The rates of [³H]-ryanodines binding to the receptors and the SR Ca²⁺ release were also promoted due to the MF exposure.It can be concluded that there is a NADH-dependent oxidase on SR membrane, which can oxidate NADH and be modulated by the two physical and chemical factors. In the process of NADH oxidation, superoxide is generated; at the meantime the RyR1 is activated.更多还原