【作者】 蔡知音；

【导师】 夏若虹；

【作者基本信息】 华东师范大学 ， 生物物理学， 2008， 硕士

【摘要】 氧化还原环境对机体细胞具有重要影响,特别是当钙释放通道的氧化还原环境发生变化时,会导致胞内的钙信号转导和钙调控机制发生变化,严重时会导致诸多疾病。肌质网（sarcoplasmic reticulum,SR）中的ryanodine receptor（RyR）是胞内重要的钙释放通道之一,它对氧化还原环境异常敏感,是氧化胁迫主要作用靶点之一。前期工作证明不同的巯基氧化剂能有效影响骨骼肌SR的钙调控机制,并发现在这些氧化剂与SR反应过程中产生了重要的氧化还原介质——超氧自由基(superoxide,O₂^·-),但它在氧化剂对SR的调控过程中的作用目前尚不明朗。本研究以提取的兔骨骼肌肌质网（SR）囊泡和钙释放通道/ryanodine receptor（RyR1）为研究对象,考察了萘醌（naphthoquinon,NQ）和硒类（selenium,Se）化合物以及伴生的O₂^·-对骨骼肌型钙释放通道（RyR1）的调控作用,并初步探讨这些氧化剂对RyR1门控的影响机制。实验利用紫外和荧光分光光度法定量检测反应伴生O₂^·-阴离子产率和通道蛋白自由巯基数量的变化。采用[³H]—ryanodine结合实验、SR囊泡钙释放动力学实验、单通道活性检测以及通道蛋白的氧化还原电势变化等实验检测了NQ类和Se类化合物对SR上RyR1活性和功能状态的影响。SDS-聚丙烯酰胺凝胶电泳和蛋白免疫印迹实验检测氧化剂导致的过氧化环境对SR上各蛋白的影响。结果发现所考察的巯基氧化剂对RyR1的活性和氧化还原电势具有双相性影响,并伴有浓度依赖性的O₂^·-产生,同时通道的自由巯基数量随氧化剂浓度增加而减少。另外,在高浓度氧化剂导致的过氧化环境下,与胞内钙转运和钙平衡机制相关的RyR1、钙泵等蛋白因巯基被氧化而发生错误交联,对蛋白的功能造成严重损伤。超氧岐化酶（superoxide dismutase,SOD）可以有效降低上述影响。推测除了氧化剂的直接氧化作用外,伴生O₂^·-在这类氧化剂调控RyR1的机制中也扮演了重要角色。超氧作为胞内信号分子,通过逐步氧化RyR1上那些对氧化环境具有不同敏感程度的自由巯基职能群,加重氧化损伤,并参与胞内钙调动和钙平衡机制的调控,这可能是在氧化胁迫环境下以蛋白质为中心的细胞损伤的分子作用基础之一。更多还原

【Abstract】 The idea that oxidative stress injures intracellular Ca²⁺mobilization and Ca²⁺ homeostasis has been widely accepted.Ryanodine receptor（RyR）is one of the most important intracellular calcium release channels and is the main target of oxidative stress.Previous studies have shown that different thiol oxidants can affect Ca²⁺ release mechanism of skeletal muscle sarcoplasmic reticulum（SR）,and the production of superoxide(O₂^·-)was detected during the reaction.To study the effect of thiol oxidants such as naphthoquinon（NQ）and selenium （Se）on the skeletal type Ca²⁺release channel/ryanodine receptor（RyR1）,generation of O₂^·-,initial rate of[³H]—ryanodine binding,release rate of Ca²⁺from SR,change of free thiols on RyR1 were detected to reveal the modulation of these reagents or O₂^·-to the channel activities.Moreover,SDS-PAGE and western blotting were used to find the effect of these oxidants on proteins in SR.The results showed that these thiol-specific oxidants could generate O₂^·-when interacting with the system.These oxidants were observed to active RyR1 at lower concentrations and to inhibit the channel at higher concentrations.Further more,the serious oxidative stress which caused by NQ or Se compounds could induce the protein cross-linking.At the same time the number of free thiols decreased with the increase in the reagents concentration.However,superoxide dismutase（SOD）could partially or completely reverse all of these effects.The results indicate that besides the directly oxidation of oxidants,O₂^·-also participates in the modulation of channel activity and the oxidation of free thiols on RyR1.It is proposed that ROS may play a role as intracellular signal molecule that participates in modulating intracellular Ca²⁺ mobility and the Ca²⁺homeostasis by oxidizing different functional free thiol groups on RyR.This is likely to underline the protein-centered molecular bases of the cellular damnification responses to an intracellular oxidative stress.更多还原