Keratan sulphate proteoglycans associated with the bone formation of embryonic chick femurs

硫酸角质素蛋白多糖与胚胎鸡股骨骨形成相关

• 批准号: 08672099
• 负责人: TAKAGI Minoru
• 金额: $ 1.22万
• 依托单位: Nihon University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08672099/
• 关键词: bone; chick embryo; glycosaminoglycan; proteoglycan; keratan sulphate; immunohistochemistry; Western blot; 骨; 鶏胚; グリコサミノグリカン; ケラタン硫酸; プロテオグリカン

The type and sistribution of sulphated proteoglycans (PGs) in the midshaft subperiosteal bone of 15-18-day embryonic chick femurs were studied immunocytochemically and biochemically, using four monoclonal antibodies (MAb 2B6, 3B3, 1B5, and 5D4). These MAb specifically recognize epitopes in chondroitin 4-sulphate (C4-S) and dermatan sulphate (DS) ; chondroitin 6-sulphate (C6-S) and unsulphated chondroitin (C0-S) ; C0-S ; and keratan sulphate (KS) respectively. Immunohistochemistry showed that staining of C4-S, DS, and KS, but not of C6-S and C0-S, was limited to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunae and bone canaliculi in 15-18-day embryonic specimens. However, no significant difference in the distribution and intensity of immunostaining was observed in these specimens. Bone proteins were extracted from fresh 18-day embryonic specimens with a three extraction procedure, 4 M guanidine HCl (GdnCl, G-1 extract), 0.4M EDTA (E-ectract), followed by GdnCl (G-2extract), to characterize mineral binding and collagenous matrix associated PGs in E-and G2-extracts respectively. Western blot analysis of E-and G2-extracts demonstrated that chondroitinase ABC-digested PGs with a molecular weight (M_r) approximately of 45,000 containing GAGs predominantly corresponding to C4-S and/or DS, with no detectable C6-S or C0-S present in the mineral and matrix phase, whereas KSPGs having an M_r of approximately 72,000 are only present in the mineral phase. These results indicate that embryonic chick bone contains small PGs having C4-S, DS, and KS chains with preferential localization to osteoid, the cell surface of osteocytes, and to the walls of osteocytic lacunac and bone canaliculi.

采用4种单克隆抗体（MAb 2B6、3B3、1B5和5D4）对15-18日龄胚鸡股骨中轴骨膜下骨中硫酸蛋白多糖（pg）的类型和分布进行了免疫细胞化学和生化研究。这些单抗特异性识别硫酸软骨素（C4-S）和硫酸皮肤素（DS）的表位；硫酸软骨素（C6-S）和未硫酸软骨素（C0-S）；C0-S;和硫酸角蛋白（KS）。免疫组化显示，C4-S、DS和KS的染色仅限于15-18天胚胎标本的类骨细胞、骨细胞表面、骨细胞腔隙壁和骨小管，而C6-S和C0-S未染色。然而，在这些标本中，免疫染色的分布和强度没有明显差异。采用4M胍HCl （GdnCl， G-1提取物）、0.4M EDTA （e -提取物）、GdnCl （g -2提取物）三种提取方法提取新鲜18天胚胎标本中的骨蛋白，分别表征e和g2提取物中矿物结合和胶原基质相关pg的特征。e -和g2提取物的Western blot分析表明，软骨素酶消化的pg分子量（M_r）约为45000，含有主要与C4-S和/或DS对应的gag，在矿物和基质相中不存在C6-S或C0-S，而M_r约为72000的KSPGs仅存在于矿物相。这些结果表明，鸡胚胎骨含有具有C4-S、DS和KS链的小PGs，它们优先定位于类骨细胞、骨细胞表面、骨细胞腔隙壁和骨小管。

项目成果

Minoru Takagi et al.: "Immunohistochemical and biochemical characterization of sulphated proteoglycans in embryonic chick bone" Journal of Nihon University School of Dentsitry. 39・3. 156-163 (1997)

Minoru Takagi 等：“胚胎鸡骨中硫酸化蛋白多糖的免疫组织化学和生物化学特征”日本大学牙科学院杂志 39・3（1997 年）。

小野世範,重政香代子,高城 稔: "鶏胚大腿骨のグリコサミノグリカンについて" 解剖学雑誌. (1997)

Yoshinori Ono、Kayoko Shigemasa、Minoru Takajo：“关于鸡胚股骨中的糖胺聚糖”解剖学杂志（1997）。