环指蛋白43对类风湿关节炎滑膜成纤维细胞基质金属蛋白酶的影响

Effects of ring finger protein 43 on matrix metalloproteinases secreted by fibroblast-like synoviocytes from rheumatoid arthritis

目的探讨环指蛋白43(RNF43)对类风湿关节炎-滑膜成纤维细胞(RA-FLS)分泌MMPs的影响。方法取膝关节置换术的RA患者滑膜组织,酶消法体外培养RA-FLS,构建RNF43慢病毒过表达质粒,转染后利用实时荧光定量(qRT)-PCR检测转染效率。转染成功后提取细胞RNA及培养液上清,分别通过qRT-PCR、ELISA检测MMP-1、MMP-3、MMP-13 mRNA及蛋白表达情况。采用独立样本t检验进行统计分析。结果当感染复数40时并加用聚凝胺可满足实验要求,RNF43的mRNA较空病毒对照组升高26158倍。与空病毒对照组相比,RNF43过表达组MMP-1、MMP-3、MMP-13的mRNA[(0.19±0.06),t=28.314,P<0.05;(0.28±0.07),t=23.413,P<0.05;(0.21±0.09),t=18.365,P<0.05]及蛋白[(31.0±9.4)pg/ml,(17.1±2.1)pg/ml,t=3.198,P=0.029]、MMP-3[(38.7±8.1)pg/ml,(24.9±3.5)pg/ml,t=3.514,P=0.015]、MMP-13[(31.0±5.4)pg/ml,(20.6±2.9)pg/ml,t=5.632,P=0.001]均明显减少。结论RNF43可能通过抑制Wnt信号通路活性对RA-FLS分泌MMPs起抑制作用。

Objective To explore the effects of ring finger protein 43(RNF43) on fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA).Methods Synovial tissues from patients with RA treated by knee arthroplasty were used to isolate FLSs by type 2 collagenase.RNF43 lentivirus overexpressing plasmid was constructed and transfected in to RA-FLS.After successful transfection,RNA and supernatant of RA-FLS were extracted.QRT-polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the mRNA and protein expression levels of matrix metalloproteinase (MMP)-1,MMP-3 and MMP-13.Data were analyzed with Student's t test.Results Transfection efficiency could meet the test requirements when the multiplicity of infection was 40 and was in conjunction with appropriate concentration of polybrene.The mRNA of RNF43 increased for 26158-fold than the control group.In vitro,compared with the control group,RNF43 could significantly inhibit the mRNA of MMP-1,MMP-3 and MMP-13 and MMP-13 [(0.19±0.06),t=28.314,P<0.05;(0.28±0.07),t=23.413,P<0.05;(0.21±0.09),t=18.365,P<0.05] and the protein of MMP-1,MMP-3 and MMP-13 and MMP-13 [(31.0±9.4) pg/ml,(17.1±2.1) pg/ml,t=3.198,P=0.029],MMP-3 [(38.7±8.1) pg/ml,(24.9±3.5) pg/ml,t=3.514,P=0.015],MMP-13 [(35.9±5.4) pg/ml,(20.6±2.9) pg/ml,t=5.632,P=0.001].Conclusion The results of study suggest that RNF43 could inhibit the secre-tion of MMPs in RA-FLS by suppressing the activity of Wnt signal pathway.