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Molecular cloning of novel osteoneurotrophins and their application as therapeutic agents for bone and cartilage diseases

新型骨神经营养蛋白的分子克隆及其在骨和软骨疾病治疗药物中的应用

基本信息

批准号：
08557098
负责人：
TAKIGAWA Masaharu
金额：
$ 12.86万
依托单位：
Okayama University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1) Neurotrophin (NT) -3 stimulated the proliferation of osteoblastic cells by increasing binding of TRE and SRE to DNA via trk C.Ascorbic acid stimulated expression of NTs, especially NT-3, in osteoblasts.2) Cultured growth cartilage cells expressed NTs and osteocytes expressed NGF and trkA is culture. Expression of NF,BDNF and NT-3 in mouse periodontal ligament cellsincreased as they became confluent while expression of trks decreased.3) Expression of trkA and trkC and NGF and NT-3 increased 2 days after fracture of mouse long bones.4) hcs-24, which was isolated from the human chondrocytic cell line HCS-2/8 as a chondrocyte- (especially hypertrophic cohondrocyte) specific gene, encoded connective tissue growth factor (CTGF). Purified CTGF and recombinant CTGF stimulated proliferation of glia cells and induced neurite formation of PC12D cells. cTGF also stimulated the proliferation and differentiation of chondrocytes, differentiation of osteoblasts, the proliferation and migration of vascular endothelial cells. These findings indicate that Hcs-24/CTGF is a novel osteochondro-neurotrophin (OCNT).5) Immunohistochemical staining with an anti-CTGF antibody revealed that spinal nerves and trigeminal ganglion were highly positive and nerves in cortex and astroglias in hippocampus were also positive. In situ hybridization revealed that motor neuron and neuron in trigeminal ganglion expressed CTGF mRNA.Carbachol stimulated neurite formation of PC12D cells.6) Two types of specific binding sites of ^<125>I-rCTGF were identified on HCS-2/8 cells. MAP kinase was found to be involved in signal transduction in the cells.7) Transgenic mice of OCNT/CTGF had skeletal disorder.8) Both IGF-I and II,which are known to induce neurite formation, stimulated expression of differentiated phenotype of chondroxytes via their respective receptors.These findings suggest that the soluble factors mentioned above can be used as therapeutic agents for bone and cartilage diseases.

1)神经营养因子(NT) -3通过trk c增加TRE和SRE与DNA的结合，刺激成骨细胞的增殖。抗坏血酸刺激成骨细胞中NT的表达，尤其是NT-3的表达。2)培养表达NTs的生长软骨细胞和表达NGF和trkA的骨细胞。小鼠牙周膜细胞中NF、BDNF和NT-3的表达随着融合而增加，trks的表达则降低。3)小鼠长骨骨折后2 d， trkA、trkC、NGF、NT-3表达升高。4) hcs-24，从人软骨细胞系HCS-2/8中分离得到的一种软骨细胞（特别是肥厚性软骨细胞）特异性基因，编码结缔组织生长因子（CTGF）。纯化CTGF和重组CTGF刺激神经胶质细胞增殖，诱导PC12D细胞形成神经突。cTGF还能促进软骨细胞的增殖分化、成骨细胞的分化、血管内皮细胞的增殖和迁移。这些发现提示Hcs-24/CTGF是一种新型骨软骨神经营养因子（OCNT）。5)抗ctgf抗体免疫组化染色显示脊髓神经和三叉神经节高度阳性，皮层神经和海马星形胶质细胞也呈阳性。原位杂交显示运动神经元和三叉神经节神经元表达CTGF mRNA。苯酚刺激PC12D细胞的神经突形成。6)在HCS-2/8细胞上鉴定出^<125>I-rCTGF的两种特异性结合位点。发现MAP激酶参与细胞的信号转导。7) OCNT/CTGF转基因小鼠存在骨骼疾病。8)已知可诱导神经突形成的IGF-I和igf - II通过各自的受体刺激软骨细胞分化表型的表达。这些结果表明，上述可溶性因子可作为骨和软骨疾病的治疗剂。