Down-Regulation of TNF-Alpha by Small Interfering RNA Inhibits Particle-Induced Inflammation In Vitro
Down-Regulation of TNF-Alpha by Small Interfering RNA Inhibits Particle-Induced Inflammation In Vitro
复制标题
小干扰 RNA 下调 TNF-α 可抑制体外颗粒诱导的炎症
DOI：
10.1111/j.1525-1594.2010.01175.x
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发表时间：
2011-07-01
影响因子：
2.4
通讯作者：
Huang, Jian-bin
中科院分区：
文献类型：
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作者：
Qin, Chu-qiang;Ding, Yue;Huang, Jian-bin
无菌性关节松动是降低关节置换术寿命的关键因素。目前除了手术翻修外，很少有有效的治疗关节松动的方法。我们探讨了肿瘤坏死因子(TNF)- α靶向小干扰RNA （siRNA）对小鼠巨噬细胞RAW264.7颗粒诱导的炎症细胞因子表达的抑制作用。化学合成靶向tnf - α的siRNA，并通过阳离子脂质体转染RAW264.7细胞。制备不同大小的陶瓷、钛和聚乙烯颗粒（0.2-1.2 μ m和1.2-10 μ m），在转染siRNA 24 h后刺激细胞。采用实时聚合酶链反应（real-time polymerase chain reaction， PCR）和酶联免疫吸附试验（ELISA）分别定量测定不同时间间隔tnf - α mRNA和蛋白水平的下调。荧光显微镜显示siRNA转染效率为85.2% +/- 3.5%。Real-time PCR结果显示，刺激后3 h，颗粒刺激+ RNA干扰（RNAi）组tnf - α mRNA水平显著低于单纯颗粒刺激组（P < 0.05）。同样，ELISA检测结果显示，转染后24 h， rnai处理组tnf - α蛋白水平显著降低（P < 0.05）。不同颗粒类型、颗粒大小刺激组间的抑制效果差异无统计学意义（P < 0.05）。我们的研究结果表明，靶向tnf - α的siRNA在不同大小的陶瓷、钛和聚乙烯颗粒刺激下可以抑制RAW264.7细胞中tnf - α的释放，并且抑制作用与颗粒的类型和大小无关。
Aseptic joint loosening is a key factor that reduces the life span of arthroplasty. There are currently few effective treatments for joint loosening except surgical revision. We explored the inhibitory effects of tumor necrosis factor (TNF)-alpha-targeted small interfering RNA (siRNA) on particle-induced inflammatory cytokine expression in the murine macrophage cell line, RAW264.7. siRNA targeting TNF-alpha was chemically synthesized and transfected into RAW264.7 cells by cationic liposomes. Ceramic, titanium, and polyethylene particles of different sizes (0.2-1.2 mu m and 1.2-10 mu m) were prepared to stimulate cells 24 h after siRNA transfection. The down-regulation of TNF-alpha mRNA and protein levels was quantitatively determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively, at different time intervals. Fluorescence microscopy showed that the siRNA transfection efficiency was 85.2% +/- 3.5%. Real-time PCR showed that the levels of TNF-alpha mRNA in the particle stimulation plus RNA interference (RNAi) groups were significantly lower compared with the particle stimulation-only groups 3 h after stimulation (P < 0.05). Similarly, ELISA essays showed that the protein levels of TNF-alpha in the RNAi-treated groups were significantly decreased 24 h after transfection (P < 0.05). There were no significant differences in the inhibitory effect among the groups stimulated by different particle types or particle sizes (P > 0.05). Our results indicated that siRNA targeting TNF-alpha can inhibit TNF-alpha release from RAW264.7 cells when stimulated by ceramic, titanium, and polyethylene particles of different sizes and that the inhibitory effects were not related to the type or size of particle.