siRNA沉默ADAM10基因对类风湿关节炎滑膜成纤维细胞凋亡的影响

Effect of ADAM10 gene silencing on apoptosis of synovial fibroblasts in rheumatoid arthritis

目的探讨小干扰RNA(siRNA)沉默解整合素金属蛋白酶(ADAM10)基因对类风湿关节炎滑膜成纤维细胞凋亡的影响。方法将ADAM10特异性siRNA转染人类风湿关节炎滑膜成纤维细胞MH7A细胞,实验分为3组,ADAM10-siRNA组转染ADAM10特异性siRNA,NC-siRNA组转染ADAM10非特异性siRNA,对照组为未经转染处理的MH7A细胞。转染48 h后采用实时荧光定量聚合酶链式反应(qRT-PCR)和蛋白免疫印迹法(Western blot)检测各组细胞中ADAM10表达水平,流式细胞仪检测细胞凋亡情况,qRT-PCR检测凋亡相关基因B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)表达水平,ELISA测定细胞培养上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-8(IL-8)含量。结果 ADAM10-siRNA组细胞中ADAM10 mRNA和蛋白表达水平较NC-siRNA组和对照组低(P <0.05)。沉默ADAM10基因后,ADAM10-siRNA组细胞凋亡率显著高于NC-siRNA组和对照组(P <0.05)。ADAM10-siRNA组细胞中Bax基因表达水平明显高于NC-siRNA组和对照组(P <0.05),Bcl-2基因表达水平明显低于NC-siRNA组和对照组(P <0.05)。ADAM10-siRNA组细胞分泌的TNF-α、IL-6和IL-8显著低于NC-siRNA组和对照组(P<0.05)。而NC-siRNA组和对照组以上各指标相比较,差异不明显(P> 0.05)。结论沉默ADAM10基因可明显促进类风湿关节炎滑膜成纤维细胞的凋亡,并进一步减少TNF-α、IL-6和IL-8等炎症因子的分泌,提示通过沉默ADAM10基因使减轻/控制类风湿关节炎炎症成为可能。

Objective To investigate the effect of ADAM10 gene silencing on apoptosis of synovial fibroblasts in rheumatoid arthritis. Methods ADAM10-specific siRNA was transfected into human rheumatoid arthritis synovial fibroblast MH7A cells. The experiment was divided into 3 groups: ADAM10-siRNA group, NC-siRNA group and control group, which were transfected with ADAM10-specific siRNA, ADAM10 non-specific siRNA or nothing, respectively. After 48 h of transfection, qRT-PCR and Western blot were used to detect the expression of ADAM10 in each group. Flow cytometry was used to detect apoptosis. The expression levels of apoptosis-related genes Bcl-2 and Bax were detected by qRT-PCR. The levels of TNF-α, IL-6 and IL-8 in the cell culture supernatant were determined by ELISA. Results The expression of ADAM10 mRNA and protein in the ADAM10-siRNA group was significantly lower than that in NC-siRNA group and control group (P < 0.05). After silencing the ADAM10 gene, the apoptosis rate of the ADAM10-siRNA group was significantly higher than that of the NC-siRNA group and the control group (P < 0.05). The expression of Bax gene in ADAM10-siRNA group was significantly higher than that in NC-siRNA group and control group (P < 0.05). The expression level of Bcl-2 gene was significantly lower than that in NC-siRNA group and control group (P < 0.05). TNF-α, IL-6 and IL-8 secreted by ADAM10-siRNA group were significantly lower than those of NC-siRNA group and control group (P < 0.05). Compared with the above indexes in the NC-siRNA group and the control group, the difference was not significant (P > 0.05). Conclusion ADAM10 gene silencing can significantly promote the apoptosis of rheumatoid arthritis synovial fibroblasts and further reduce inflammatory factors such as TNF-α, IL-6 and IL-8, suggesting that silencing of ADAM10 gene makes it possible to reduce/control rheumatoid arthritis inflammation.