B7-1双价抗体对自身免疫性狼疮样肾病的免疫干预效应研究

Autoimmune diseases is characterized by a global loss of self-tolerance with activation of hyper-reactive T and B cells leading to production of pathogenic autoantibodies and tissue injury, Among more than 30 kinds of autoimmune disease, systemic lupus erythematosus (SLE) is representative. Despite substantial research on SLE, the etiology and pathogenesis are still not well understood. Due to these obstacles, the disease treatment has lagged behind many other diseases, seldom therapies were specific and high effectual. Only one new drug for lupus was approved by the United States Food and Drug Administration (FDA) in the last 50 years. As a promising kind of therapy on SLE, infusion with monoclonal antibodies or other specific proteins that specifically inhibit or interfere with T cell and/or B cell hyper-reaction is revolutionizing the treatment of autoimmune disorders. The steps of our investigation were: 1. To develop the animal model of SLE. Intraperitoneal injection of the hydrocarbon oil pristane into C57BL/J6 (B6) mice leads to lupus-like autoimmune syndrome, This model is well suited to investigate the pathogenesis process and clinic manifestation of SLE as human-beings. 2. To treat the lupus-like model by anti-B7-1 diabody (only containing two VH and VL fragments), which was generated by our lab, in order to suppress the APC-T -B cell signal pathway.? ??3. To investigate the therapeutic effect of B7-1 diabody, the serology of BUN, autoantibody, antinuclear antibody, evaluation of renal disease(proteinuria, glomerulonephritis score, immune-complexes deposition) were dynamic monitored. 4. To study the molecular mechanism of the autoantibody generation and activation by analyzing the immunology indexes of lupus-like disease model systematically, including cytokines, cell apoptosis, immune cell activation, immune cell function, and the dynamic changing process of NF-κB acting as a very important transcription factor for the transduction signal pathways.

自身免疫性疾病是由自身反应性T及B细胞异常活化所致。涉及疾病30余种。发病机制尚未明确。若能特异性阻断或削弱T、B细胞异常应答即可发挥治疗作用。本课题旨在运用自行研制的基因工程抗人B7-1双价抗体可同时识别人与小鼠B7-1分子的特性。从事以下研究：1.采用化学法（pristane）建立类拟人类系统性红斑狼疮(SLE)发病过程及临床表现的小鼠（C57BL/J6 (B6)）狼疮样肾病模型为研究切入点；2.运用B7-1双价抗体序贯治疗，以阻抑APC-T-B信号轴；3.通过血清学（血尿素氮、自身抗体、抗核抗体）及肾脏病理损伤评价（蛋白尿、肾小球肾炎评分、免疫复合物等）指标的动态监测，考察抗体的治疗效应；4.通过系统分析疾病模型及抗体干预前、后免疫学指标（细胞因子、细胞凋亡、免疫细胞活化及功能）及信号转导通路中重要转录因子NF-κB的动态变化，探究自身抗原产生及活化的分子机制。

自身免疫性疾病是由自身反应性T细胞及B细胞异常活化所致的疾病，发病机制目前尚不明确。但已有的研究表明，若能特异性阻断或削弱T细胞及B细胞异常应答即可发挥治疗作用。本课题运用自行研制的基因工程抗人B7-1抗体可同时识别人与小鼠B7-1分子的特性。进行了下述的研究。采用化学法（pristane）先行建立类拟人类系统性红斑狼疮(SLE)发病过程及临床表现的小鼠狼疮样肾炎模型为研究切入点，运用B7抗体序贯治疗，以阻抑APC-T-B信号轴；通过血清学（血尿素氮、自身抗体、抗核抗体）及肾脏病理损伤评价（蛋白尿、肾小球肾炎评分、免疫复合物等）指标的动态监测，考察抗体的治疗效应；在对小动物研究的基础上，运用化学法，再行建立模拟人类因环境因素所致的非人灵长类动物食蟹猴的自身免疫性狼疮样肾炎模型，再行运用抗体进行干预后对治疗效应及可能的分子机制进行客观的评价及探讨；通过系统分析疾病模型及抗体干预前、后免疫学指标（细胞因子、细胞凋亡、免疫细胞活化及功能）、血清学指标（血尿素氮、自身抗体、抗核抗体）及肾脏病理损伤评价（蛋白尿、肾小球肾炎评分、免疫复合物等）的分析，探究特异性的抗体类生物制剂在自身免疫性疾病的犯病机制研究及治疗中的意义。取得的重要成果有：成功构建了分泌抗B7-1及 B7-2抗体的工程细胞株；先行建立的小鼠（C57BL/J6 (B6）自身免疫性狼疮样肾炎模型，通过特异性抗体干预后，疾病模型的病理改变得到明显改善，包括免疫细胞的活化降低，自身抗体的产生减少，肾功能得到改善等。进一步对大型非人灵长类动物食蟹猴的相应疾病模型的抗体干预研究，得到了一致的研究结果。提示特异性抗体阻断APC向T细胞提供共刺激信号，可抑制T细胞的活化及其对 B细胞应答的辅助作用，进而减少自身抗体的产生及由此产生的病理损伤。对临床此类疾病具有潜在的治疗意义。