靶向肿瘤坏死因子-α慢病毒介导RNA干扰的实验性治疗研究

The experimental gene therapy with lentiviral-mediated RNA interference targeting tumor necrosis factor-α

目的：观察靶向小鼠TNF-α RNA干扰（RNAi）慢病毒载体颗粒对小鼠巨噬细胞株RAW264.7表达TNF-α、IL-1β及IL-6的影响，及对胶原诱导性关节炎（CIA）的治疗作用。方法分别用靶向小鼠TNF-α RNAi慢病毒颗粒、慢病毒阴性对照、空白对照感染小鼠巨噬细胞株 RAW264.7，检测TNF-α、IL-1β、IL-6 mRNA表达水平及TNF-α蛋白水平。制作小鼠CIA模型，设慢病毒RNAi治疗组、慢病毒阴性对照、空白对照和阳性对照，分别尾静脉注射 RNAi慢病毒颗粒、慢病毒阴性对照、PBS溶液，腹腔注射甲氨蝶呤，观测CIA关节炎积分，检测小鼠TNF-α血清水平，病理检查受累关节组织。结果①慢病毒治疗组TNF-αmRNA表达水平为0.291±0.021，低于慢病毒阴性对照0.925±0.013，差异有统计学意义（t=25.4，P〈0.01），抑制率为68.5%；②慢病毒治疗组TNF-α血清水平为（249±11） ng/ml，低于阴性对照（382±6） ng/ml，差异有统计学意义（t=10.31，P〈0.05），抑制率为34.7%。③慢病毒治疗组、阴性对照及空白对照之间，IL-1β、IL-6 mRNA表达水平差异无统计学意义（t=1.00，1.22，P均〉0.05）。④尾静脉注射后第8天，慢病毒治疗组关节炎分值为2.50±0.19，低于空白对照3.63±0.18及阴性对照3.75±0.16，差异均具统计学意义（F=42.8，P〈0.01），慢病毒治疗组与阳性对照的关节炎分值缓慢下降，至少持续至尾静脉注射后2周。⑤慢病毒治疗组、阳性对照TNF-α血清水平分别为（35±6） pg/ml、（32±7） pg/ml，均低于阴性对照47±3，差异有统计学意义（t=3.03，4.11，P均〈0.01）。病理显示慢病毒治疗组关节炎症细胞浸润减轻。结论靶向小鼠TNF-αRNAi在体外、体内有效抑制TNF-αmRNA及蛋白表达，降低CIA关节炎分值，减轻炎症细胞浸润。慢病毒介导RNAi为RA治疗提供一可行、有效方法。

Objective To investigate the effects of lentiviral-mediated RNA interference （RNAi） targeting tumor necrosis factor-α（TNF）-αgene on the expression of TNF-α, interleukin （IL）-1β, IL-6 of murine macrophages RAW264.7, and the efficiency of RNAi experimental gene therapy for the murine collagen-induced arthritis （CIA）. Methods The RAW264.7 macrophages were infected by lentivirus-RNAi particles, then stimulated by Lipopolysaccharides （LPS）. The TNF-α, IL-1β, IL-6 expression of RAW264.7 macrophages were measured with real-time polymerase chain reaction （PCR） and enzyme linked immunosorbent assay （ELISA）. CIA models were esta-blished in DBA/1 mice using bovine type Ⅱ collagen. The treatment effect of lentivirus-RNAi on CIA were observed through arthritis scores, serum TNF-α measurement and hind paw paraffin section hematoxylin/eosin staining after lentivirus-RNAi particles tail vein injection. Results The TNF-αmRNA relative expression level of lentiviral RNAi group was 0.291 ±0.021, significantly lower than that of negative control group 0.925±0.013 （t=25.4, P〈0.01）. The inhibition rate in mRNA levels was 68.5%. The serum TNF-α level of lentiviral RNAi group was [（249 ±11） ng/ml], significantly lower than that of negative control [（382±6） ng/ml] （t=10.31, P〈0.05）. The inhibition rate of protein levels was 34.7%. It had no effect on the IL-1β and IL-6 mRNA expression. On the 8th day after systemic administration, the arthritis score of＆amp;nbsp;lentivirus-RNAi group was 2.50±0.19, which was significantly lower than that of blank controls （3.63 ±0.18） and negative controls （3.75 ±0.16） （F=42.8, P〈0.01）. From now on, arthritis score of lentivirus-RNAi group and positive control decreased slowly to at least 2 weeks after treatment induction. The serum TNF-α levels of lentivirus-RNAi group and positive controls were [（35±6） pg/ml] and [（32±7） pg/ml] significantly lower than that of negative controls [（47±3） pg/ml] （t=3.03, 4.11, P〈0.01） respectively. Morphological examination showed that the lentivirus-RNAi decreased CIA pathological manifestations. Conclusion Lentiviral-mediated RNAi targeting murine TNF-α gene can effectively inhibit TNF-α expression both in vitro and in vivo, which also effectively improve the CIA arthritis score. Lentiviral-mediated RNAi targeting TNF-αgene provides a potential strategy for rheumatoid arthritis （RA） treatment.