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.

软骨组织的形成引发了软骨骨形成。在形成软骨霉菌后不久，霉菌中心的软骨细胞变成肥厚的和钙化。与钙化软骨区域的新血管形成同时，将骨细胞前体募集到骨化中心，在该中心，分化的破骨细胞逐渐将软骨逐渐替换为骨骼并挖掘骨髓腔。因此，钙化的软骨必须是在胚胎发育过程中出现分化的破骨细胞时出现拳头的地方。在这里，我们检查了钙化软骨可能在形成分化破骨细胞的功能作用的可能性。我们从年轻的兔子中隔离了板状软骨细胞，并进行了培养。在第6天（在汇合阶段），第22天（在成熟阶段），第42天（在肥厚阶段），第48天（在钙化的早期阶段）和第57天（分别在钙化的阶段）。通过坑形成测定（骨最小17：347-359,1992）和陷阱测定法（血液74：1295-1302,1989）评估培养基对破骨碎屑分化的刺激活性。结果清楚地表明，钙化的软骨细胞分泌了破骨细胞分化的因子刺激性。然后，我们发现了发育骨骼的胎儿雌性骨膜软骨的鸟选择素提取物中类似的破骨细胞分化因子。纯化了活性原理，并发现与软骨瘤蛋白-II（CHM-II）相同。 CHM-II在3-10 ng/ml的剂量rage处显着刺激了破骨细胞分化，但不需要维生素D_3的存在进行作用。