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Purification of Osteoclast Differentiation Factor produced by Epiphyseal Cartilage

骨骺软骨产生的破骨细胞分化因子的纯化

基本信息

批准号：
07672014
负责人：
HIRAKI Yuji
金额：
$ 1.47万
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：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Endochondral bone formation is initiated by the formation of cartilage tissue. Soon after the cartilaginous mold is formed, chondrocytes in the central part of the mold become hypertrophic and calcified. Concomitantly with neovascularization in the calcified cartilage zone, the bone-cell precursors are recruited to the ossification center where the differentiated osteoclasts gradually replace cartilage into bone and excavate the bone marrow cavity. Therefore, calcified cartilage must be the place where the differentiated osteoclasts appear for the fist time during embryonic development. Here we examined a possibility that calcified cartilage may play a functional role for formation of the differentiated osteoclasts. We isolated grwoth-plate chondrocytes from young rabbits, and cultured. The culture media were conditioned for two days each on Day 6 (at the confluent stage), Day 22 (at the maturing stage), Day 42 (at the hypertrophic stage), Day 48 (at the early calcified stage), and Day 57 (at the heavily calcified stage), respectively. Stimulatory activity of the medium on osteoclastic differentiation was assessed by pit formation assay (Bone Min.17 : 347-359,1992) and TRAP assay (Blood 74 : 1295-1302,1989). The results clearly indicated that calcified chondrocytes secreted a factor stimulatory for osteoclastic differentiation. Then we found the similar osteoclast differentiation factor in the guanidine extracts of fetal bovine epiphyseal cartilage of developing bone. The active principle was purified, and found identical with chondromodulin-II (ChM-II). ChM-II markedly stimulated osteoclastic differentiation at the dose rage of 3-10 ng/ml, but it did not required the presence of vitamin D_3 for action.

软骨内骨形成是由软骨组织的形成开始的。软骨霉菌形成后不久，霉菌中心部分的软骨细胞变得肥大和钙化。伴随着钙化软骨区的新血管形成，骨细胞前体被募集到骨化中心，在那里分化的破骨细胞逐渐将软骨替换成骨并挖掘骨髓腔。因此，在胚胎发育过程中，钙化软骨一定是分化的破骨细胞首次出现的地方。在这里，我们研究了一种可能性，钙化软骨可能发挥功能的作用，形成分化的破骨细胞。分离培养幼兔骺板软骨细胞。分别在第6天（在汇合阶段）、第22天（在成熟阶段）、第42天（在肥大阶段）、第48天（在早期钙化阶段）和第57天（在重度钙化阶段）将培养基调节两天。通过小坑形成试验（Bone Min.17：347- 359，1992）和TRAP试验（Blood 74：1295- 1302，1989）评估培养基对骨细胞分化的刺激活性。结果清楚地表明，钙化的软骨细胞分泌一种刺激骨细胞分化的因子。在胎牛骨骺软骨胍提取物中发现了类似的破骨细胞分化因子。活性成分进行纯化，发现与软骨调节素-II（ChM-II）相同。ChM-Ⅱ在3-10 ng/ml剂量范围内明显促进成骨细胞分化，但不需要维生素D_3的存在。