小干扰RNA对人类风湿关节炎患者滑膜细胞基因表达的影响  

Effect of small interfering RNA on gene expression of synovial cells in patients with rheumatoid arthritis

作　　者：侯春凤[1] 孙闵[2] 李树杰[1] 赵建宏[1] 王吉波[3] 

机构地区：[1]济宁市第一人民医院风湿免疫科,山东省济宁市272002 [2]济宁医学院临床学院,山东省济宁市272013 [3]青岛大学附属医院风湿免疫科,山东省青岛市266071

出　　处：《中国组织工程研究》2013年第46期8062-8068,共7页Chinese Journal of Tissue Engineering Research

摘　　要：背景:类风湿关节炎的病因尚不十分明确,但核转录因子κB在类风湿性关节炎发病中的作用,己逐渐引起风湿病学者的关注。目的:通过RNA干扰技术阻断人类风湿关节炎滑膜细胞核转录因子κB信号通路,探索其在类风湿关节炎治疗中的应用前景。方法:分离、消化、培养滑膜细胞备用。根据小分子干扰RNA设计原则,确定核转录因子κB的小分子干扰RNA四条目标基因序列,合成并构建核转录因子κB小分子干扰RNA表达载体。将构建好的4条pGenesil-1/核转录因子κB小分子干扰RNA表达载体转染入生长状态良好的一代滑膜细胞,并设立空白和阴性对照组。收集转染后12 h、24 h、48 h、72 h、5 d、7 d不同时间段的细胞,并提取RNA。测定细胞内核转录因子κB mRNA相对表达水平,筛选出有效抑制核转录因子κB mRNA表达的小分子干扰RNA质粒载体。结果与结论:核转录因子κB在人类风湿关节炎滑膜细胞高表达,3#pGenesil-1/核转录因子κB能显著抑制人类风湿关节炎滑膜细胞核转录因子κB mRNA表达。RNA干扰技术可阻断核转录因子κB mRNA的表达,因此,可将阻断核转录因子κB信号通路作为基因治疗类风湿关节炎的靶点。BACKGROUND： The etiological factor for rheumatoid arthritis remains unclear, but the effects of nuclear factor-KB on the onset of rheumatoid arthritis have been gradually paid great attention by rheumatologists. OBJECTIVE： By using the RNA interference （RNAi） technique to block the signal pathway of nuclear factor-KB mRNA of human rheumatoid arthritis synovial cells, this study explored its application prospect in the treatment of rheumatoid arthritis. METHODS： The synovial cells were isolated, digested, and cultured for further use. In accordance with the design principle of small interfering RNA （siRNA）, target sequences of siRNA of nuclear factor-KB were identified, and siRNA expression vector of nuclear factor-KB was synthesized and constructed, The four pGenesil-l/nuclear factor-KB siRNA expression vectors were transfected into the first passage of synovial cells that well grew. Blank and negative control groups were set. Cells at 12, 24, 48, 72 hours, 5 and 7 days after transfection were collected, and RNA was extracted. Intracellular nuclear factor-KB mRNA expression levels were measured, and siRNA plasmid vector that could effectively inhibit nuclear factor-KB mRNA expression was screened out. RESULTS AND CONCLUSION： Nuclear factor-KB highly expressed in synovial cells after human rheumatoid arthritis. 3#pGenesil-l/nuclear factor-KB apparently suppressed nuclear factor-KB mRNA expression in synovial cells with human rheumatoid arthritis. RNAi technique blocked nuclear factor-KB mRNA expression. Therefore, the block of nuclear factor-KB signal pathway might be a good target for rheumatoid arthritis gene therapy.

关 键 词：组织构建 组织构建细胞学实验 关节炎 类风湿 核转录因子ΚB pGenesil-1表达载体 RNA干扰 实时荧光定量聚合酶链反应 

分 类 号：R318[医药卫生—生物医学工程]