siRNA抑制survivin基因表达对类风湿关节炎滑膜成纤维细胞增殖与凋亡的影响

siRNA inhibition of survivin gene expression in rheumatoid arthritis synovial fibroblast proliferation and apoptosis

目的研究靶向存活素(survivin)的小干扰RNA(small interfering RNA,siRNA)抑制survivin基因表达对类风湿关节炎滑膜成纤维细胞(rheumatoid arthritis synovial fibroblasts,RASFs)增殖与凋亡的影响。方法体外分离培养RA患者滑膜成纤维细胞(RASFs),设计合成靶向survivin的siRNA及阴性对照,用脂质体法转染RASFs细胞;实时定量聚合酶链反应和蛋白印迹法检测RASFs中survivin的mRNA表达及蛋白水平。四甲基偶氮唑蓝法检测细胞增殖;TUNEL法检测细胞凋亡。结果实验组与阴性对照siRNA组及空白对照组相比,转染48h后滑膜成纤维细胞survivin的mRNA及蛋白表达水平明显下降(P<0.05)。实验组与阴性对照siRNA组及空白对照组相比,转染后的滑膜成纤维细胞增殖能力明显下降(P<0.05)。实验组转染后24h、48h、72h生长抑制率分别为(11.5±2.6)%、(26.2±3.4)%、(47.6±4.1)%,于转染后72小时最为显著。实验组凋亡率为(23.87±1.6)%,显著高于阴性对照组(9.72±1.15)%和空白对照组(8.70±1.09)%(均P<0.05)。结论靶向survivin的siRNA通过下调survivin表达水平,抑制滑膜成纤维细胞增殖并促进其凋亡。

Objective To study the targeting survivin small interfering RNA（ siRNA） to inhibit proliferation and apoptosis survivin gene expression in rheumatoid arthritis synovial fibroblasts（ RASFs）. Methods RA patients were isolated and cultured in vitro synovial fibroblasts（ RASFs）,designed and synthesized siRNA targeting survivin and negative control,by liposome transfection RASFs cell; real-time quantitative polymerase chain reaction（ PCR） and Western blot RASFs detect mRNA expression and protein levels of survivin. Tetrazolium blue（ MTT） assay of cell proliferation;TUNEL assay apoptosis. Results The experimental group compared with the negative control siRNA group and control group,48 h after transfection of synovial fibroblasts survivin mRNA and protein expression levels were significantly decreased（ P〈0. 05）. The experimental group compared with the negative control siRNA group and control group,synovial fibroblast proliferation after transfection significantly decreased（ P〈0. 05）. After the experimental group transfected 24 h,48h,72 h growth inhibition rates were（ 11. 5 ± 2. 6） %,（ 26. 2 ± 3. 4） %,（ 47. 6 ± 4. 1） %,at 72 hours after transfection most significant. The rate of apoptosis in experimental group（ 23. 87 ± 1. 6） %,significantly higher than the negative control group（ 9. 72 ±1. 15） % and the control group（ 8. 70 ± 1. 09） %（ all P〈0. 05）. Conclusion siRNA targeting survivin expression levels through reducing survivin,inhibit synovial fibroblast proliferation and promotes apoptosis.