模拟失重条件下硫氧还蛋白1对神经母细胞瘤细胞的微丝骨架保护作用研究  

Study of the Protection Role of Thioredoxin1 on Actin Cystoskeleton in Neuroblastoma Cells under Simulated Microgravity

作　　者：李琦[1] 王晓刚[1] 凌树宽[1] 李莹辉[1] 李英贤[1] 

机构地区：[1]中国航天员科研训练中心,航天医学基础与应用国家重点实验室,北京100094

出　　处：《航天医学与医学工程》2013年第6期450-454,共5页Space Medicine & Medical Engineering

基　　金：留学回国启动基金项目

摘　　要：目的研究回转模拟失重条件下硫氧还蛋白1(thioredoxin1,Trx1)对神经母细胞瘤细胞SH-SY5Y微丝骨架的保护效应及其分子机制。方法转染Trx1入SH-SY5Y细胞,采用回转方法模拟失重,采用免疫荧光化学方法分析细胞微丝骨架的形态和结构变化,采用DCF-DA荧光探针检测细胞内活性氧(reactive oxygen species,ROS)水平;利用AMS修饰结合聚丙烯酰胺凝胶电泳检测微丝骨架蛋白半胱氨酸残基的氧化还原状态;使用超速离心法分离细胞内球状肌动蛋白(global actin,G-actin)单体和纤丝状肌动蛋白(filamentous actin,F-actin)聚合体,Western blot方法鉴定F-actin及G-actin的水平。结果回转模拟失重引起SH-SY5Y细胞微丝骨架排列紊乱,呈弥散不规则分布,Trx1可以保护模拟失重环境导致的微丝骨架的紊乱;回转引起细胞内ROS升高达到对照组2.8倍,而过表达Trx1的细胞回转后ROS的产生仅为对照组的1.7倍;回转模拟失重导致细胞微丝骨架半胱氨酸残基被氧化,而Trx1可以部分阻止半胱氨酸残基氧化;回转模拟失重后SH-SY5Y细胞F-actin发生解聚,G-actin增加,Trx1能够部分对抗F-actin的解聚,维持F-actin/G-actin比率。结论 Trx1可能通过降低细胞内ROS水平,抑制微丝骨架蛋白半胱氨酸残基的氧化,对模拟失重状态下肌动蛋白的解聚与聚合起到重要的调控作用。Objectiv e To investigate the protection effect and the underlying molecular mechanism of thioredox- inl （Trxl） on actin cytoskeleton in neuroblastoma （SH-SY5Y） cells with clinostat simulated microgravity. Methods Trxl was transfected into SH-SY5Y cells. Clinostat was adopted to simulate weightlessness. Cell im- munofluorescence technique was applied to assay the shape and structure of actin cytoskeleton. DCF-DA fluo- rescent probes were employed to detect the level of reactive oxygen species （ROS） in the intracellular. Modi- fied polyacrylamide gel electrophoresis method with AMS was used to detect the redox state of protein cysteine residues in the cytoskeleton of actin protein. Uhracentrifugation was used to seperate G-actin monomer and F- actin polymer in the intracellular. The levels of F-actin and G-actin were evaluated with western blotting. Re- suits The actin cytoskeleton arranged in disorder and diffused irregularly after rotation, while Trxl could coun- teract the destruction of microfilament cytoskeleton induced by simulated weightlessness. Compared with the control group, the level of intracellular ROS increased to 2.8 times after rotation, whereas the cellular level of ROS after over expression with Trxl increased merely to 1.7 times ; Amount of actin proteins were oxidized af- ter rotation, however Trxl protected cysteine residues from oxidation partially; Intracellular F-actin were depo- lymerized to G-actin after rotation, while Trxl could partially rescue the depolymerization of F-actin and stabi- lize the F-actin/G-actin ratio. Conclusions Trxl may reduce the level of intracellular ROS, partially keep cys- teine in reduced state and inhibit the depolymerization of F-actin. It may play an important regulating role to keep the integrity of both morphology and function of depolymerization and polymerization of the actin cytoskel- eton under simulated microgravity.

关 键 词：模拟失重 Trx1 微丝骨架 氧化应激 氧化还原调节 

分 类 号：R852.22[医药卫生—航空、航天与航海医学] R854[医药卫生—临床医学]