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Prevention of joint contracture following joint immobilization using gene therapy's method

利用基因疗法预防关节固定后的关节挛缩

基本信息

批准号：
13671507
负责人：
HASHIMOTO Jun
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

Joint contracture followed by prolonged joint immobilization is a serious problem in orthopaedic treatment. The cause of that was reported as that proliferation of fibrous synovial tissue in the joint cavity was characteristic. To prevent the contracture followed by immobilization, we planned to suppress the proliferation of synoviocytes by gene transfer of transcription factor E2F decoy into synoviocytes. As the animal model of joint immobilization, we used Wilson's rat model (Wilson et al Clin Orthop 1988). At 1, 2, 3 and 4 weeks after joint immobilization, we examined the Hematoxylin and eosin staining of the sagittal section of knee joint and confirmed the proliferation of synovial tissue and adhesion of extracellular matrix in the joint reproductively. In vitro study, we examined the semi-quantitative transfection efficiency of gene transfer into fibroblast-like cell line (3Y1-B and NRK) and primary generated rat synoviocyte using FITC-labeled oligonucleotide (FITC ODN) included in HVJ-envelope vector, and determined the optimal condition of gene transfer. In vivo, we observed the high efficient gene transfer into synovial tissue by intraarficular injection of FITC ODM included in HVJ envelope vector, and almost no transfer into other tissue such as articular cartilage, meniscus and ligament. Now we examine in vivo gene transfer using E2F decoy included in HVJ envelope and analyze the proliferation of synovial tissue and production of extracellular matrix by molecular biologically and histologically.

关节挛缩后的长期关节制动是一个严重的问题，在骨科治疗。据报告，其原因是关节腔内的纤维性滑膜组织增生是特征性的。为了防止挛缩，随后制动，我们计划抑制滑膜细胞的增殖，通过基因转移的转录因子E2 F诱饵到滑膜细胞。作为关节固定的动物模型，我们使用Wilson大鼠模型（Wilson等Clin Orthop 1988）。分别于制动后1、2、3、4周行膝关节矢状面苏木精-伊红染色，观察关节滑膜组织增生和细胞外基质粘附情况。在体外实验中，我们检测了HVJ-包膜载体中的FITC标记寡核苷酸（FITC ODN）对成纤维细胞样细胞系3 Y1-B和NRK以及原代大鼠滑膜细胞的半定量转染效率，并确定了基因转染的最佳条件。在体内实验中，我们观察到通过关节腔内注射HVJ包膜载体中包含的FITC ODM，基因高效地转移到滑膜组织中，并且几乎没有转移到其他组织如关节软骨、半月板和韧带中。本研究利用HVJ包膜中的E2 F诱饵进行体内基因转移，并从分子生物学和组织学角度分析滑膜组织的增殖和细胞外基质的产生。