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

我们先前建立了一种使用仙台病毒/HVj在体内关节内注射的转染方法。在这项研究中，我们构建并转染基因到体内动物关节炎模型以及体外从类风湿性关节炎（RA）患者获得的人滑膜成纤维细胞中，在此基础上，我们建立了一种新的基因转染方法，并在动物模型中研究了与创伤愈合相关的基因转染的体内效果。在关节炎模型中的炎症作用我们构建了针对转录因子NF κ B的“诱饵”，NF κ B与炎性细胞因子的产生和粘附分子的表达密切相关。其在CIA大鼠的关节内转染显示关节炎的等级，足肿胀和关节炎指数显着改善，也显着抑制关节破坏的组织学发现。接下来，我们构建了质粒DNA编码p21位于p53的下游，其突变已多次报告在RA。p21也被认为与细胞凋亡。通过HVJ脂质体转染RA患者滑膜成纤维细胞，体外实验显示该基因可诱导细胞凋亡，并显著抑制细胞增殖.韧带愈合模型我们建立了一种通过动脉内递送HVJ-脂质体直接转染大鼠髌韧带的新方法。此外，我们将编码肝细胞生长因子（HGF）的质粒DNA转染到大鼠愈合的髌韧带中，观察到I型胶原的表达，其纤维平行排列，类似于正常韧带。

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.