Establishment of in vivo direct gene transfer system into joint using HVJ-liposome

我们之前在体内建立了仙台病毒/HVj关节内注射转染方法。在这项研究中，我们构建并转染基因到动物体内关节炎模型和体外获得的类风湿性关节炎（RA）患者滑膜成纤维细胞中，以寻找RA基因治疗的可能性。此外，我们在动物模型中实现了一种新的转染方法，并研究了转染与伤口愈合有关的基因在体内的作用。我们构建了一个针对转录因子NFkB的“诱饵”，NFkB与炎症细胞因子的产生和粘附分子的表达密切相关。CIA大鼠关节内转染其可显著改善其关节炎等级、足跖肿胀和关节炎指数，并明显抑制关节破坏的组织学表现。接下来，我们构建了编码p21的质粒DNA，该质粒位于p53的下游，其突变已在RA中多次报道。P21也被认为与细胞凋亡有关。用hvj脂质体将该基因转染到RA患者的滑膜成纤维细胞中，在体外显示细胞凋亡并明显抑制细胞增殖2。我们建立了一种动脉给药hvj脂质体直接转染大鼠髌骨韧带的新方法。此外，我们将编码肝细胞生长因子（HGF）的质粒DNA转染到愈合的大鼠髌骨韧带中，观察到纤维平行排列的I型胶原蛋白的表达，与正常韧带相似。

We previously established a transfection method of intra-articular injection using Sendai virus/HVj in vivo. In this study we constructed and transfected genes into an animal arthritis model in vivo as well as into human synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) in vitro, in search of possibility of gene therapy for RA.Furthermore we achieved a novel transfection method into ligaments in animal models and investigated in vivo effect of transfection of a gene relating to wound healing.I.Anti-inflammatory effect in an arthritis modelWe constructed a "decoy" against transcription factor, NFkB which closely associated with production of inflammatory cytokines and expression of adhesion molecules. Its intra-articular transfection in CIA rats showed significant improvement of arthritic grades, paw swelling and arthritis index, and also markedly suppressed histological findings of joint destruction.Next we constructed plasmid DNA encoding p21 locating downstream of p53, the mutation of which had repeatedly been reported in RA.p21 is also thought to associate with apoptosis. The transfection of the gene into human synovial fibroblasts from RA patients by use of HVJ-liposomes demonstrated apoptosis of the cells as well as marked suppression of cell proliferation in vitro.2. Ligament healing modelWe established a novel method of direct transfection in rat patellar ligaments by intra-arterial delivery of HVJ-liposomes. Moreover we transfected plasmid DNA encoding hepatocyte growth factor (HGF) into healing patellar ligament in rats and observed the expression of collagen type I with parallel alignment of fibers, which resembled normal ligaments.