Wang Hai-jun, Yu Chang-long, Kishi Hiroyuki, Motoki Kazumi, Mao Ze-bin, Muraguchi Atsushi
Institute of Sports Medicine, Peking University Third Hospital, Beijing 100083, China.
Chin Med J (Engl). 2006 Aug 20;119(16):1365-73.
Osteoarthritis (OA) is a chronic and incurable disease, lacking effective treatment. Gene therapy offers a radical different approach to the treatment of arthritis. Even though the etiology of OA remains unclear, there is now considerable evidence to suggest that interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) are the main mediators in the pathogenesis of OA. The goal of this study was to determine the efficacy of local expression of interleukin-1 receptor antagonist (IL-1Ra) and soluble tumor necrosis factor-alpha receptor type I (sTNF-RI) by direct adenoviral-mediated intra-articular gene delivery in the rabbit model of osteoarthritis.
Adenoviral vectors containing IL-1Ra or sTNF-RI genes were constructed. OA was induced in both hind knees of 12 New Zealand white rabbits by the excision of the medial collateral ligament plus medial meniscectomy. Five days after surgery, approximately 1 x 10(8) plaque-forming units (pfu) of adenovirus were injected into the joint space of the knee through the patellar tendon. A total of 12 operated rabbits were divided into four groups. Three experimental rabbit groups received 1 x 10(8) pfu of adenovirus encoding either IL-1Ra (3 rabbits), sTNF-RI (3 rabbits) or IL-1Ra and sTNF-RI in combination (3 rabbits), into both knee joints respectively. An inflamed control group of 3 rabbits received approximately 1 x 10(8) pfu of Ad-GFP into both joints. Three days after injection of the adenovirus, both knees of each rabbit were lavaged with 1 ml of saline solution through the patellar tendon. At day 7, the rabbits were sacrificed, and the knees were lavaged, dissected and analyzed for effects of transgene expression. Levels of IL-1Ra and sTNF-RI expression in recovered lavage fluids were measured using a cytokine ELISA kit. Cartilage from the lesion areas of medial femoral condyle and synovium were fixed, embedded, sectioned and stained with hematoxylin and eosin (cartilage and synovium) and toluidine blue (cartilage). The samples were examined by light microscopy and quantitatively evaluated.
Intra-articular delivery of IL-1Ra resulted in a significant inhibition of cartilage degradation, but did not affect synovial changes. In contrast, rabbit knee joints receiving sTNF-RI alone showed no detectable reduction in cartilage degradation. However, double gene transfer of IL-1Ra and sTNF-RI resulted in a higher suppression of the cartilage degradation and an observable reduction in synovitis. These data add to and confirm that IL-1Ra has good chondroprotective properties, but TNF-alpha blockade has little effect on joint destruction.
The enhanced therapeutic effects of both antagonists in combination suggest inhibition of multiple inflammatory cytokines may be more efficacious than blockade of either cytokine alone in treating OA.
骨关节炎（OA）是一种慢性且无法治愈的疾病，缺乏有效的治疗方法。基因治疗为关节炎的治疗提供了一种截然不同的方法。尽管OA的病因仍不明确，但现在有大量证据表明白细胞介素-1（IL-1）和肿瘤坏死因子-α（TNF-α）是OA发病机制中的主要介质。本研究的目的是通过直接腺病毒介导的关节内基因递送，在兔骨关节炎模型中确定白细胞介素-1受体拮抗剂（IL-1Ra）和可溶性肿瘤坏死因子-α受体I型（sTNF-RI）局部表达的疗效。
构建了含有IL-1Ra或sTNF-RI基因的腺病毒载体。通过切除内侧副韧带加内侧半月板切除术，在12只新西兰白兔的双后膝关节诱导骨关节炎。术后5天，通过髌腱将约1×10⁸ 噬斑形成单位（pfu）的腺病毒注入膝关节腔。总共12只手术兔分为四组。三个实验兔组分别将1×10⁸ pfu编码IL-1Ra（3只兔）、sTNF-RI（3只兔）或IL-1Ra和sTNF-RI组合（3只兔）的腺病毒注入双膝关节。3只兔的炎症对照组双关节分别接受约1×10⁸ pfu的Ad-GFP。腺病毒注射后3天，通过髌腱用1 ml盐溶液冲洗每只兔的双膝关节。在第7天，处死兔子，冲洗、解剖膝关节并分析转基因表达的效果。使用细胞因子ELISA试剂盒测量回收的灌洗液中IL-1Ra和sTNF-RI的表达水平。将股骨内侧髁病变区域的软骨和滑膜固定、包埋、切片，并用苏木精和伊红（软骨和滑膜）以及甲苯胺蓝（软骨）染色。通过光学显微镜检查样本并进行定量评估。
关节内递送IL-1Ra可显著抑制软骨降解，但不影响滑膜变化。相比之下，单独接受sTNF-RI的兔膝关节软骨降解未见明显减少。然而，IL-1Ra和sTNF-RI的双基因转移导致对软骨降解的更高抑制以及滑膜炎的明显减轻。这些数据补充并证实了IL-1Ra具有良好的软骨保护特性，但TNF-α阻断对关节破坏几乎没有影响。
两种拮抗剂联合使用增强的治疗效果表明，在治疗OA时，抑制多种炎性细胞因子可能比单独阻断任何一种细胞因子更有效。
