NFκB寡核苷酸诱骗剂诱导耐受性DC对大鼠实验性类风湿性关节炎干预作用

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by the influxation of synovia and synovial compartments with immune cells including dendritic cells (DCs). Tolerogenic DCs (tolDCs) were characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability.As a promising immunotherapeutic strategy for RA, TolDCs have received increasing attention. The activation of nuclear factor-kappa B (NF-κB) plays a key role in the maturation of DC. Oligodeoxynucleotide decoy(ODN Decoy), a recently developed genetic method, can completely block transcription factor function.Previous studies showed that NF-κB ODN Decoy was imported into the cells to prevent the activation of NF-κB. In our previous study,the tolDC was successfully constructed through NF-κB Decoy ODN method towards isolated spleen cells. This tolDC was characterized in vitro by a stable immature phonotype and a potent immunosuppressive ability. Our preliminary data also showed that collagen induced arthritis (CIA) rats were treated with these NF-κB ODN Decoy -pretreated DCs loaded with Bovine TypeⅡCollagen antigen. Arthritis severity was evaluated by arthritic score and joint histopathology. Therefore, we plan to explore the underlying mechanism of the therapeutic ability of these tolDCs against CIA. The study will provide the experimental basis and theoretical foundation for the potential therapeutic utility of Decoy-induced to DCs in rheumatoide arthritis.

未成熟树突状细胞(dendritic cell,DC)诱导特异性免疫耐受应用于自身免疫病的治疗日益受到关注，应用NF-κB 寡核苷酸诱骗剂（NF-κB ODN Decoy）可有效阻断DC细胞内NF-κB活性，从而抑制DC的表型和功能成熟，进而可能成为新的自身免疫病干预手段。我们前期已用NF-κB ODN Decoy处理获得了大鼠耐受性DC,并将之负载牛Ⅱ型胶原（BⅡC），初步显示了对大鼠实验性风湿性关节炎具有一定确切的防治作用。本项目旨在进一步观察该策略对实验性类风湿性关节炎的治疗效果，并深入探讨该耐受性DC可否在大鼠体内形成稳定的“微嵌合”，及其对T淋巴细胞活化及内在的信号转导的影响，从细胞及分子水平探讨BⅡC-Decoy DC 对类风湿性关节炎的干预机制，为该策略应用于类风湿性关节炎的临床治疗提供更有力的实验证据。

未成熟树突状细胞(dendritic cell,DC)诱导特异性免疫耐受应用于自身免疫病的治疗日益受到关注，应用NF-κB 寡核苷酸诱骗剂（NF-κB ODN Decoy）可有效阻断DC细胞内NF-κB活性，从而抑制DC的表型和功能成熟，进而可能成为新的自身免疫病干预手段。本项目组用NF-κB ODN Decoy处理获得了大鼠耐受性DC，并将之负载牛Ⅱ型胶原（BⅡC），以此耐受性DC输注入实验性类风湿性关节炎(collagen induced arthritis，CIA)大鼠体内，观察了对大鼠CIA的干预效果，探讨该耐受性DC 对T淋巴细胞活化的影响，从细胞及分子水平探讨BⅡC-Decoy DC 对类风湿性关节炎的干预机制。研究发现来源于病程中CIA大鼠和正常大鼠并经NF-κ B ODN Decoy处理和负载BⅡC的DC分别对病程中自身CIA大鼠和同种异体CIA大鼠有良好的治疗效果，使其症状、体征、滑膜组织病理表现均得到一定程度的逆转与改善。同时我们发现负载了BⅡC的耐受性DC具有抗原特异性，对无关抗原无反应，不会引起广泛的免疫抑制，且对大鼠没有骨髓、肝肾相关副作用，证明了耐受性DC具有较好的安全性。另外，耐受性DC的干预明显改变了大鼠体内T细胞亚群分布及细胞因子分泌模式：分析Th1（CD3+CD8-IFN-γ+）、Th2（CD3+CD8-IL-4+）、Th17（CD3+CD8-IL17+）和Treg（CD3+CD8-Foxp3+），显示Th1、Th17占比减少，Th2、Treg占比增加，促炎细胞因子IFN-γ、IL-2减少而抗炎细胞因子IL-4和IL-10增加。RA病人血清及CIA大鼠关节滑膜组织中20S蛋白酶体活性及底物蛋白IκBα的表达增高。耐受性DC通过改变T细胞和细胞因子的表达从而缓解大鼠关节炎。在干预机制的研究中，我们还发现，耐受性DC能够在大鼠体内形成较长时间的微嵌合，小动物活体成像显示，耐受性DC在输注后的第35天仍能检测到信号，提示外源性输注的耐受性DC在大鼠体内可能形成稳定的“微嵌合”状态，成为免疫干预的机制之一。综上所述，本项目研究证明用NF-κB 寡核苷酸诱骗剂构建的耐受性DC对大鼠实验性类风湿性关节炎有很好的干预效果，可作为临床类风湿性关节炎的临床治疗策略作进一步探讨。本项目研究结果总结发表论文5篇，培养硕士2名，达到既定指标，申请结题。