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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。
Objective To investigate the protective effect of thioredoxin 1 (Trx1) on the actin cytoskeleton of neuroblastoma SH-SY5Y under rotational simulated weightlessness and its molecular mechanism. Methods Trx1 was transfected into SH-SY5Y cells. The weight loss was simulated by rotary method. The morphological and structural changes of actin cytoskeleton were analyzed by immunofluorescence staining. The DCF-DA fluorescent probe was used to detect the intracellular reactive oxygen species (ROS) ) Levels were measured. The redox status of the cysteine residues in actin cytoskeleton was detected by AMS and polyacrylamide gel electrophoresis. The cells of global actin (G-actin) were separated by ultracentrifugation And filamentous actinomycetes, F-actin aggregates. The levels of F-actin and G-actin were identified by Western blot. Results Rotational simulated weightlessness caused the skeletal microvessel structure of SH-SY5Y cells to disordered and dispersed irregularly. Trx1 could protect the microfilament skeleton caused by simulated weightlessness environment. Rotation induced intracellular ROS up to 2.8 times that of control group Trx1-producing cells produced only 1.7-fold more ROS than the control group; rotational mimic weight loss resulted in the oxidation of cysteine residues in the actin cytoskeleton, whereas Trx1 partially blocked the oxidation of cysteine residues; After F-actin disaggregated and G-actin increased in SH-SY5Y cells, Trx1 partially antagonized depolymerization of F-actin and maintained the F-actin / G-actin ratio. Conclusion Trx1 may play an important role in the depolymerization and polymerization of actin in simulated weightlessness by decreasing intracellular ROS level and inhibiting the oxidation of cysteine residues in actin.