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

我们先前建立了仙台病毒/HVJ体内关节内注射的转染法。在这项研究中，我们构建并将基因导入体内的动物关节炎模型和体外获得的类风湿关节炎(RA)患者的滑膜成纤维细胞中，以寻找RA的基因治疗的可能性。此外，我们还实现了一种新的动物模型韧带中的转基因方法，并研究了体内转导与伤口愈合相关的基因的效果。I.关节炎模型的抗炎作用我们构建了一个与炎症细胞因子的产生和黏附分子的表达密切相关的转录因子诱饵NFkB。在CIA大鼠的关节内转染p21基因后，关节炎分级、足肿胀和关节炎指数明显改善，关节破坏的组织学表现也明显受到抑制。接下来，我们构建了位于p53下游的编码p21的DNA，该突变在RA.p21中已多次报道。p21的突变也被认为与细胞凋亡有关。结论：1.通过脂质体将该基因导入RA患者的滑膜成纤维细胞，体外培养的人滑膜成纤维细胞出现明显的细胞凋亡和细胞增殖抑制。韧带愈合模型我们建立了一种新的方法，通过动脉注射hvj-脂质体的方法将hvj-脂质体直接导入大鼠膝关节韧带。此外，我们还将编码肝细胞生长因子(HGF)的质粒DNA导入大鼠愈合的膝关节韧带中，观察到与正常韧带相似的平行排列的I型胶原的表达。

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.