双特异性磷酸酶8通过与促分裂原活化蛋白激酶1相互作用调控类风湿关节炎成纤维样滑膜细胞增殖 凋亡  

Dual specificity phosphatases 8 directly interacts with mitogen-activated protein kinase 1 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells

作　　者：张锌[1] 南鹤[1] 李爽[2] Zhang Xin;Nan He;Li Shuang(Department of Rheumatology and Immunology,China-Japan Union Hospital of Jilin University,Changchun 130033,China;Four Department of Neurology,China-Japan Union Hospital of Jilin University,Changchun 130033,China)

机构地区：[1]吉林大学中日联谊医院风湿免疫科,长春130033 [2]吉林大学中日联谊医院神经内四科,长春130033

出　　处：《中华风湿病学杂志》2022年第7期433-438,I0002,共7页Chinese Journal of Rheumatology

基　　金：吉林省自然科学基金自由探索重点项目(NO.2020122316JC)。

摘　　要：目的:探讨RA-成纤维样滑膜细胞(FLS)中双特异性磷酸酶8(DUSP8)DUSP8与促分裂原活化蛋白激酶(MAPK)1之间的相互作用,及其对RA-FLSs增殖、凋亡的影响。方法:培养RA-FLS和正常FLS,采用实时荧光定量聚合酶链式反应(RT-qPCR)、蛋白质印迹法检测两组细胞DUSP8 mRNA表达水平。采用细胞转染技术及RNA干扰技术,构建DUSP8过表达细胞系及DUSP8沉默细胞系。CCK-8法检测各组细胞增殖,流式细胞术检测各组细胞凋亡。蛋白质印迹法检测各组细胞DUSP8、MAPK1、p-MAPK1、ki-67、Bax蛋白表达水平。间接免疫荧光实验分析DUSP8与MAPK1的空间共定位,免疫共沉淀实验分析DUSP8与MAPK1蛋白之间是否存在相互作用。计量资料两组间比较采用独立样本 t检验,多组间比较采用单因素方差分析,进一步两两比较采用LSD- t检验。 结果:DUSP8在RA-FLS mRNA[(2.4±0.6)和(11.2±0.8), t=21.63, P<0.001]和蛋白表达[(0.24±0.04)和(0.74±0.08), t=9.45, P<0.001]水平均显著低于FLS。与空白对照组和过表达对照组相比,在RA-FLS细胞转染pcDNA3.1-Myc-DUSP8可显著抑制RA-FLS细胞的增殖[(90.5±5.6),(92.5±1.8),(56.4±4.4), F=138.60, P<0.001],增加细胞凋亡率[(12.7±1.4)%和(12.6±1.3)%和(27.5±3.0)%, F=16.98, P<0.001],上调细胞的DUSP8'[(0.49±0.05),(0.45±0.04)和(0.73±0.07)]、Bax[(0.39±0.06),(0.36±0.05)和(0.89±0.10)]蛋白表达水平,下调ki-67[(1.07±0.12)和(1.11±0.16)和(0.70±0.08)]、p-MAPK1/MAPK1[(0.59±0.06)和(0.65±0.07)和(0.39±0.03)]蛋白表达水平(均 P<0.001);与空白对照组和沉默对照组相比,在RA-FLS细胞转染siRNA-DUSP8可显著促进RA-FLS细胞的增殖[(90.5±5.6)和(91.1±2.9)和(128.3±4.6), F=137.50, P<0.001],降低细胞凋亡率[(12.7±1.4)%和(13.2±1.2)%和(5.4±0.7)%, F=16.98, P<0.001],下调细胞中的DUSP8[(0.492±0.048)和(0.432±0.051)和(0.102±0.024)]、Bax[(0.391±0.062)和(0.411±0.058)和(0.090±0.011)]蛋白表达水平,上调ki-67[(1.07±0.12)和(1.11±0.15)和(1.93±0.22)]、p-MAObjective To explore the interaction between dual specificity phosphatases 8(DUSP8)and mitogen-activated protein kinase 1(MAPK1)in rheumatoid arthritis fibroblast-like synovial(RA-FLS),and its effect on the proliferation and apoptosis of RA-FLSs.Methods RA-FLS and normal fibroblast-like synovial cells(FLS)were cultured.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)and Western blotting were used to detect the expression levels of DUSP8 mRNA and protein in the two groups of cells.DUSP8 overexpression cell lines and DUSP8 silencing cell lines were constructed using cell transfection technology and RNA interference technology,respectively.Cell counting kit 8(CCK-8)method was used to detect cell proliferation in each group,and flow cytometry was used to detect cell apoptosis in each group.Western blotting was used to detect the expression levels of DUSP8,MAPK1,p-MAPK1,ki-67 and Bax protein in each group.The indirect immunofluorescence experiment was used to analyze the spatial co-localization of DUSP8 and MAPK1,and the co-immunoprecipitation experiment was used to analyze whether there was interaction between DUSP8 and MAPK1 protein.The t-test was used to compare the means of the two groups.One-way analysis of variance was used to compare the mean values of the three groups of samples,and then the LSD-t tests were used to compare the two groups.Results In RA-FLS,both mRNA[(2.4±0.6)vs(11.2±0.8),t=21.63,P<0.001]and protein levels[(0.24±0.04)vs(0.74±0.08),t=9.45,P<0.001]of DUSP8 were significantly lower than FLS.Compared with the blank control group and the overexpression control group,RA-FLS cells transfected with pcDNA3.1-Myc-DUSP8 could inhibit the proliferation of RA-FLS cells[(90.5±5.6)vs(92.5±1.8)vs(56.4±4.4),F=138.60,P<0.001],increase the rate of apoptosis significantly[(12.7±1.4)%vs(12.6±1.3)%vs(27.5±3.0)%,F=16.98,P<0.001],increase the expression levels of DUSP8[(0.49±0.05)vs(0.45±0.04)vs(0.73±0.07),Bax(0.39±0.06)vs(0.36±0.05)vs(0.89±0.10)]and down-regulate the expression le

关 键 词：关节炎 类风湿 细胞增殖 细胞凋亡 双特异性磷酸酶8 促分裂原活化蛋白激酶1 

分 类 号：R593.22[医药卫生—内科学]