类风湿性关节炎成纤维样滑膜(FLS)细胞中DDR2相互作用分子的筛选鉴定及其对FLS细胞侵袭能力的影响

Effect of DDR2 Interaction Protein Vimentin in the Invasion of Rheumatoid Arthritis Fibroblast-Like Synovial Cells

目的:在类风湿性关节炎(RA)成纤维样滑膜(FLS)细胞中筛选并鉴定盘状结构域受体2(DDR2)的相互作用蛋白,并研究其对FLS细胞侵袭能力的影响。方法:首先利用免疫沉淀结合SDS-PAGE分离鉴定DDR2互作蛋白波形蛋白,进而采用免疫沉淀和激光共聚焦实验,进一步验证波形蛋白是DDR2的相互作用蛋白,最后采用Transwell实验考察波形蛋白对RA FLS细胞侵袭能力的影响。结果:Ⅱ型胶原刺激引起RA FLS细胞中DDR2的磷酸化水平升高,DDR2被活化,活化前后共有8个DDR2相互作用蛋白发生变化,经质谱分析,其中变化最大的是波形蛋白和膜联蛋白A2;在HEK293T细胞中,免疫共沉淀结果显示Fc DDR2与波形蛋白存在相互作用;激光共聚焦结果显示在RA FLS细胞中,DDR2和波形蛋白存在共定位;下调RA FLS细胞中波形蛋白的表达后,细胞的侵袭能力较对照组显著下降。结论:波形蛋白是DDR2的相互作用蛋白,Ⅱ型胶原可引起RA FLS细胞的DDR2磷酸化水平升高,并通过波形蛋白促进RA FLS细胞的侵袭。

Objective： To screen the discoidin domain receptor 2（DDR2） interaction protein in rheumatoid arthritis（RA） fibroblast-like synovial（FLS） cell and investigate the interaction protein function on invasion of RA FLScells.Methods： Firstly,we used immunoprecipitation combined with SDS-PAGE to screen DDR2 interaction pro-tein.Secondly,Co-IP and confocal microscopy were used to further confirm the protein vimentin.Thirdly,tran-swell assay was used to evaluate the effect of vimentin on invasion of RA FLS cells.Results： Collagen Ⅱ couldenhance the phosphorylation of DDR2 in RA FLS cells,and we found 8 DDR2 interaction protein after DDR2 ac-tivation,vimentin had been chosen to do the further study.Co-IP and confocal microscopy results confirmed theinteraction between DDR2 and vimentin in HEK293 T and FLS cells.After down-regulate expression of vimentin,the invasion of RA FLS cells reduced remarkably.Conclusion： Vimentin is the interaction protein of DDR2,colla-gen Ⅱ could enhance the phosphorylation of DDR2 in RA FLS cells,and encourage the invasion of FLS cellsthrough vimentin.