TNF-α对类风湿性关节炎患者滑膜成纤维细胞RUNX3表达的影响及意义

摘要

目的 探讨TNF-α对类风湿性关节炎患者滑膜成纤维细胞RUNX3表达的影响及意义.方法 从类风湿性关节炎患者滑膜组织中分离并培养原代成纤维细胞,加入不同浓度(0、0.1、1、10、50 ng/mL)TNF-α分别作用0、3、6、12、24 h,采用实时荧光定量PCR法检测RUNX3 mRNA相对表达量.取传3～8代成纤维细胞,随机分为RUNX3过表达组和对照组,分别感染人源RUNX3腺病毒、绿色荧光蛋白(GFP)对照腺病毒,感染24 h两组均加入50 ng/mL TNF-α再刺激24 h;采用实时荧光定量PCR法检测两组炎性因子[IL-6、前列腺素E2(PGE-2)、趋化因子CXC基序配体9(CXCL-9)、基质金属蛋白酶2(MMP-2)及MMP-13]表达,采用Transwell侵袭实验检测细胞侵袭能力.取传3～8代成纤维细胞,随机分为RUNX3沉默组和对照组,分别采用脂质体转染法转染siRUNX3、NC-control siRNA,转染24 h两组均加入50 ng/mL TNF-α再刺激24 h;采用实时荧光定量PCR法检测IL-6、PGE-2、CXCL-9、MMP-2及MMP-13 mRNA表达.结果 成纤维细胞RUNX3 mRNA相对表达量随着TNF-α浓度升高及作用时间延长而逐渐升高,呈浓度和时间依懒性,以TNF-α浓度50 ng/mL、作用时间24 h时RUNX3 mRNA相对表达量上调最为显著(P均<0.05).RUNX3过表达组和对照组穿膜细胞数分别为(56±4)、(20±3)个,两组比较P<0.01.RUNX3过表达组IL-6、PGE-2、MMP-13 mRNA相对表达量均高于对照组(P<0.05或<0.01),两组CXCL-9、MMP-2 mRNA相对表达量比较P均>0.05.RUNX3沉默组IL-6、CXCL-9、MMP-2及MMP-13 mRNA相对表达量均低于对照组(P均<0.05),两组PGE-2 mRNA相对表达量比较P>0.05.结论 TNF-α可促进成纤维细胞RUNX3表达,并呈浓度和时间依赖性;RUNX3表达升高可通过调控IL-6、MMP-13表达而促进成纤维细胞的侵袭能力.%Objective To explore the effect of TNF-αon the expression of RUNX3 in synovial fibroblasts in patients with rheumatoid arthritis and its mechanism .Methods RASFs were isolated and primary cultured from synovial tissues of patients with rheumatoid arthritis , TNF-αat different concentrations (0, 0.1, 1, 10, 50 ng/mL) was added for 0, 3, 6, 12, and 24 hours respectively , and the relative expression of RUNX 3 mRNA was detected by quantitative real-time PCR. The fibroblasts of the 3rd-8th generations were randomly divided into the RUNX 3 overexpression group and the NC control group, which were infected with the human RUNX 3 adenovirus and the green fluorescent protein ( GFP) control adenovi-rus, respectively, and after 24 h, both groups were treated with 50 ng/mL TNF-αfor 24 h.The real-time fluorescent quan-titative PCR was used to detect the expression of inflammatory factors [interleukin-6 (IL-6), prostaglandin E2 (PGE-2), chemokine CXC-9 ligand, matrix metalloproteinase 2 (MMP-2), and MMP-13] in the two groups.Transwell assay was used to detect cell invasive ability .The fibroblasts of the 3rd-8th generations were randomly divided into the RUNX 3 silen-cing group and NC control group , which were transfected with siRUNX 3 and NC control siRNA by liposome transfection method, respectively, and after 24 h, both groups were treated with 50 ng/mL TNF-αfor 24 h; the real-time fluorescent quantitative PCR was used to detect the expression of IL-6, PGE-2, CXCL-9, MMP-2, and MMP-13.Results The rela-tive expression level of RUNX 3 mRNA in fibroblasts increased gradually with the increase of TNF-αconcentrations and the duration of action , with a dose-and time-dependent manner;the up-regulation of the relative expression of RUNX 3 mRNA was the most significant when TNF-αconcentration was 50 ng/mL and the treatment time was 24 h (all P<0.05).The number of transmembrane cells in the RUNX3 overexpression group and the NC control group was 56 ±4 and 20 ±3, re-spectively (P<0.01).The relative expression of IL-6, PGE-2 and MMP-13 mRNA in the RUNX3 overexpression group was higher than that in the NC control group (P<0.05 or P<0.01).There was no significant difference in the relative ex-pression of CXCL-9 and MMP-2 mRNA between the two groups (P>0.05).The relative expression of IL-6, CXCL-9, MMP-2 and MMP-13 mRNA in the RUNX3 silencing group was lower than that in the control group (P<0.05).There was no significant difference in the relative expression of PGE-2 mRNA between the two groups ( P>0.05).Conclusion TNF-αcan promote the expression of RUNX3 in fibroblasts with a dose-and time-dependent manner, and the increased expressionof RUNX3 can promote the invasion of fibroblasts by regulating the expression of IL -6 and MMP-13.