目的:观察核不均一核糖核蛋白(hnRNP)K的表达与定位以及亚砷酸钠(NaAsO2)刺激NIH3T3细胞的反应情况及氧化应激变化,探讨JNK信号通路在hnRNP K蛋白聚集体形成中的作用.方法:构建重组载体pcDNA3-hnRNP K-HA,转染NIH3T3细胞,荧光显微镜观察内源性hnRNP K在细胞中的表达与定位,以及在NaAsO2不同刺激时间该蛋白聚集体的形成情况,并利用活性氧(ROS)检测试剂盒测定胞内ROS水平；给予JNK、MEK、PI3K/Akt、NF-κB和核转运等5种信号通路的抑制剂预处理后,观察聚集体的变化情况.结果:重组质粒构建正确,荧光显微镜观察显示hnRNP K主要定位于胞核中,而重组质粒转染NIH3T3细胞后,可见胞内有蛋白聚集体形成并随NaAsO2刺激时间的延长而递增,且过表达hnRNP K可抑制NaAsO2刺激细胞生成ROS; JNK信号通路抑制剂SP600125明显抑制聚集体的形成.结论:hnRNP K主要定位在NIH3T3细胞的胞核中,胞浆少量分布；NaAsO2可诱导NIH3T3细胞形成hnRNP K蛋白聚集体,此聚集体可抑制胞内ROS生成,且该聚集体的形成有赖于JNK介导的信号通路.%AIM:To explore the role of JNK signaling pathway in the formation of heterogeneous nuclear ribonucleoprotein (hnRNP) K protein aggregates by observing the expression and localization of hnRNP K in NIH3T3 cells induced by sodium arsenite (NaAsO2).METHODS:The recombinant vector pcDNA3-hnRNP K-HA was constructed and transfected into NIH3T3 cells.The expression and localization of endogenous hnRNP K and distribution of the protein aggregates stimulated with NaAsO2 at different time points were observed under fluorescence microscope.The level of reactive oxygen species (ROS) in the cells was detected by a ROS assay kit.After pretreated with the inhibitors of 5 signaling pathways (JNK,MEK,PI3K/Akt,NF-κB and nuclear transport),the changes of the protein aggregates were observed.RESULTS:The recombinant plasmid was correctly constructed.The hnRNP K was mainly distributed in the nucleus.The intracellular fluorescent aggregates increased with the prolonging stimulation of NaAsO2 in the cells transfected with the recombinant plasmid,and the overexpression of hnRNP K inhibited ROS generation in the cells induced by NaAsO2.SP600125,an inhibitor of JNK signaling pathway,significantly inhibited the formation of protein aggregates.CONCLU-SION:The hnRNP K is primarily located in the nucleus of NIH3T3 cells,with a small amount in the cytoplasm.The formation of protein aggregates is significant after NaAsO2 stimulation,which can restrain the level of intracellular ROS,and the process is involved in JNK signaling pathway.
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