摘要
目的探讨CCR5基因沉默对类风湿关节炎(RA)大鼠滑膜细胞炎症因子TNF-α、IL-6、MMP-1及MMP-3的影响。方法将SPF级Wistar雄鼠饲养一周后随机分为Control(WC)组与RA模型组,采用胶原诱导法构建RA大鼠模型,然后分离和培养RA大鼠滑膜细胞,再将细胞分为4组:RA组(RA大鼠滑膜细胞)、RA+NC组(加入脂质体包裹的NC siRNA)、RA+mock组(加入脂质体)、RA+siRNA组(加入脂质体包裹的CCR5 siRNA)。利用RNA干扰技术干扰大鼠滑膜细胞CCR5的表达,干扰后用qRT-PCR检测各组滑膜细胞CCR5及炎症因子TNF-α、IL-6、MMP-1及MMP-3 mRNA相对表达水平,用Western Blot检测其CCR5蛋白表达情况,并用ELISA方法检测各组滑膜细胞上清液中TNF-α、IL-6、MMP-1、MMP-3的水平。结果干扰大鼠滑膜细胞CCR5的表达后,与RA组比较,RA+siRNA组CCR5 mRNA及TNF-α、IL-6、MMP-1、MMP-3 mRNA及蛋白水平均显著降低(P<0.05),而RA+NC组、RA+mock比较组差异无统计学意义(P>0.05)。结论沉默CCR5可抑制CCR5 mRNA及其蛋白的表达,并有助于降低RA大鼠滑膜细胞的炎症因子TNF-α、IL-6、MMP-1及MMP-3的表达,减轻大鼠滑膜细胞的炎症反应,并为以后利用RNA干扰技术沉默CCR5用于RA的治疗提供新的方向。
Objective To investigate the effect of CCR5 gene silencing on the TNF-α,IL-6,MMP-1 and MMP-3 inflammation factor of rheumatoid arthritis(RA)rats.Methods The RA rat model was constructed by collagen induction method.RA rat synovial cells were isolated and cultured,and the cells were divided into 4 groups:RA group(RA rat synovial cells),RA+NC group(adding liposome-encapsulated NC siRNA),RA+mock group(adding liposome),RA+siRNA group(adding liposome-encapsulated CCR5 siRNA).RNA interference technology was used to interfere with the expression of CCR5 in rat synovial cells.After interference,qRT-PCR was used to detect the relative expression levels of CCR5 and inflammatory factors TNF-α,IL-6,MMP-1 and MMP-3 mRNA in synovial cells of each group.Western Blot was used to detect the expression of CCR5 protein,and ELISA was used to detect the levels of TNF-α,IL-6,MMP-1,and MMP-3 in the supernatant of synovial cells in each group.Results After interfering with the expression of CCR5 in rat synovial cells,compared with the RA group,the mRNA and protein levels of CCR5,TNF-α,IL-6,MMP-1,MMP-3 in the RA+siRNA group were significantly reduced(P<0.05).Compared with RA group,there was no significant difference between RA+NC group and RA+mock group(P>0.05).Conclusion Silencing CCR5 can inhibit the expression of CCR5 mRNA and its protein,and help to reduce the expression of TNF-α,IL-6,MMP-1 and MMP-3 in RA rat synoviocytes,and reduce inflammation in rat synovial cells.This research provides a new direction for using RNA interference technology to silence CCR5 for the treatment of RA.
作者
王友强
兰由玉
李世勇
黄晓玲
WANG Youqiang;LAN Youyu;LI Shiyong;HUANG Xiaoling(Department of Laboratory Medicine,The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China;Department of Rheumatology and Immunology,The Affiliated Hospital of Southwest Medical University,Luzhou 646000,Sichuan,China)
出处
《西部医学》
2021年第9期1300-1304,1310,共6页
Medical Journal of West China
基金
西南医科大学校级课题(2019ZQN038)。
