靶向IL-6基因慢病毒介导RNA干扰治疗鼠胶原诱导性关节炎的实验性研究

IL-6 plays an important role in the pathogenesis of rheumatoid arthritis, blocking the IL-6-mediated inflammatory signaling pathway can relieve joint inflammation reaction and improve the prognosis. Currently biologics IL-6 receptor antagonist tocilizumab injection made significant efficacy in clinical. In this study，by Liposome-mediated chemical synthesis of siRNA transfecting of mouse macrophage cell line Raw264.7, we observe its effect on macrophage expression and secretion of cytokines such as IL-6 using Real-Time PCR and ELISA. We designed shRNA taking use of screening siRNA fragments, constructed lentiviral vector, and packaged into a lentivirus particles,then immunized DBA / 1 mice with bovine collagen type Ⅱ to produce CIA, trying through intravenous injection targeting IL-6 shRNA lentivirus , assessmented lentiviral vector-mediated IL-6 RNA interference therapeutic effect of collagen-induced arthritis by CIA arthritis improvement of arthritis score and joint inflammation tissue infiltration. We may explore a new treatment techniques for curing rheumatoid arthritis in this way.

白介素6在类风湿关节炎发病机制中起重要作用,通过阻断IL-6介导的炎症信号通路可阻断关节炎症反应改善预后。目前生物制剂IL-6受体的人源化单克隆抗体托珠单抗注射液在临床中取得显著疗效。本研究应用脂质体介导化学合成siRNA的方法转染小鼠巨噬细胞株Raw264.7，采用Real-Time PCR和ELISA方法观察其对巨噬细胞表达和分泌IL-6等细胞因子的影响。利用经过筛选的siRNA片段设计成shRNA，构建表达shRNA的慢病毒载体，并将其包装成慢病毒颗粒。用牛Ⅱ型胶原免疫DBA /1小鼠制作CIA，尝试使用CIA尾静脉注射表达靶向IL-6 shRNA慢病毒的方法，通过CIA关节炎分值及关节炎症组织浸润改善情况评价慢病毒载体介导的IL-6 RNA干扰对胶原诱导性关节炎的治疗作用，探索类风湿关节炎的一种新的治疗技术。

目的：探讨靶向小鼠白细胞介素-6（IL-6）基因RNA干扰慢病毒载体颗粒（LV-IL6-RNAi）对小鼠巨噬细胞株J774A.1细胞IL-6表达的影响，及其对胶原诱导性关节炎（CIA）的治疗作用。方法：设计合成3对特异性针对小鼠IL-6基因的小干扰RNA（small interfering RNA, siRNA）序列，筛选高效siRNA序列按慢病毒载体构建要求，构建高效LV-IL6-RNAi。LV-IL6-RNAi感染小鼠巨噬细胞株J774A.1细胞，用实时定量聚合酶链反应（RT-qPCR）检测J774A.1细胞的IL-6、肿瘤坏死因子-α（TNF-α）和IL-1β mRNA相对表达水平，以检测LV-IL6-RNAi体外干扰效率。构建胶原诱导性关节炎模型（CIA），设LV-IL6-RNAi组、LV-NC-RNAi组、甲氨蝶呤（MTX）阳性对照组、PBS空白对照组，分别关节腔注射LV-IL6-RNAi、LV-NC-RNAi、腹腔注射MTX、PBS溶液，观测CIA小鼠关节炎评分，ELISA检测小鼠IL-6、IL-1β和TNF-α血清水平，组织病理检测炎症关节的炎性细胞浸润数量。结果：成功构建了高效LV-IL6-RNAi。在体外J774A.1细胞水平上，LV-IL6-RNAi组IL-6 mRNA相对表达水平显著性低于LV-NC-RNAi组、培养基空白对照组。首次免疫注射后第28-35天成模，建模率为96.43％。首次免疫注射后第35天，CIA-LV-IL6-RNAi组关节炎评分开始降低，首次免疫注射后第42天，CIA-LV-IL6-RNAi组关节炎评分显著性低于CIA-LV-NC-RNAi、CIA-PBS组。CIA-LV-IL-6-RNAi组IL-6、IL-1β及TNF-α血清水平显著性低于CIA-PBS组、CIA-LV-NC-RNAi组；CIA-LV-IL6-RNAi组和CIA-MTX 组炎症关节病理切片高倍镜下炎症细胞的浸润数量均显著性少于CIA-LV-NC-RNAi组和CIA-PBS组。结论：成功构建的高效LV-IL6-RNAi，可作为类风湿关节炎（RA）实验性基因治疗的有效手段。