Immunocytochemical study on the behavior and function of osteoclasis using monoclonal antibodies to osteoclasts

使用破骨细胞单克隆抗体对破骨行为和功能进行免疫细胞化学研究

• 批准号: 03670896
• 负责人: AKAMINE Akifumi
• 金额: $ 1.34万
• 依托单位: Kyusyu University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670896/
• 关键词: bone resorption; osteoclast; osteoblast; immunohistochemistry; sialoglycoprotein; monoclonal antibody; osteopontin; リソゾーム性膜シアロ糖タンパク質; モノクロ-ナル抗体

Osteoclasts are primary cells responsible for bone resorption. However, the precise mechanism as to osteoclastic bone resorption remains unclear. We therefore examined immunocytochemically the behavior and the function of osteoclasts and osteoblasts in bone rmodeling.1.Immunocytochemical localization of a major lysosomal membrane sialoglycoprotein (LGP 107) in osteoclasts : The immunocytochemical localization was investigated of LGP 107 using rat osteoclasts at various stages of differentiation. LGP 107 was exclusively confined to the apical plasma mambrane at the ruffled border of the active osteoclasts. The protein was also concentrated in a number of endocytic vacuoles near the ruffled border membrane. However, the post and/or resting osteoclasts were totally devoid of the membraneous localization of LGP 107. These results indicate that the protein is largely synthesized in the active osteoclast and rapidly translocated to the ruffled border membrane. LGP 107 is suggested to contrib … More ute to the formation and maintenance of the specialized acidic environment for bone reorption.2.Expression and localization of LGP 107 in osteoblastic lineage cells : The immunocytochemical localization of LGP 107 was investigated in osteoblast linage cells involved in osteoclastic bone resorption. Strong immunoreaction products for LGP 107 occurred on the plasma membranes in the osteoblasts and osteocytes prior to the appearance of osteoclasts. Furthermore, strong reactions were also observed on the plasma membranes in the osteoblastic cells adjacent to the active osteoclasts. These data suggest that LGP 107 in osteoblastic cells and osteocytes may play an important role in cell-recognition and/or cell-adhesion, and that LGP 107 may be involved in osteoblastic degradation of the osteoid as well as exposure of the bone surface.3.Production of monoclonal antibodies to osteoclasts by in vitro immunization : The characteristics of a monoclonal antibody produced against produced against osteoclast-like multinucleated cells (MNC_S) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. After the in vitro immunization was performed, the monoclonal antibody HOK1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNC_S and their putative migratory traces on culture dishes. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK1 was osteopontin. Postive HOK1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNC_S. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts might trap this protein on their cell surface. Less

破骨细胞是负责骨吸收的主要细胞。然而，骨钙素骨吸收的确切机制仍不清楚。因此，我们研究了破骨细胞和成骨细胞在骨重建中的行为和功能的免疫细胞化学。1.破骨细胞中主要溶酶体膜唾液酸糖蛋白（LGP 107）的免疫细胞化学定位：利用大鼠破骨细胞在不同分化阶段研究了LGP 107的免疫细胞化学定位。LGP 107仅限于活性破骨细胞皱褶边缘的顶端质膜。蛋白质也集中在皱褶边缘膜附近的许多内吞空泡中。然而，后和/或休息破骨细胞完全缺乏LGP 107的膜定位。这些结果表明，蛋白质主要是在活性破骨细胞中合成，并迅速转运到皱褶缘膜。建议LGP 107为 ...更多信息 LGP 107在成骨细胞系中的表达和定位：采用免疫细胞化学方法，对LGP 107在参与骨吸收的成骨细胞系中的定位进行了研究。在破骨细胞出现之前，成骨细胞和骨细胞的质膜上出现LGP 107的强免疫反应产物。此外，在与活跃的破骨细胞相邻的成骨细胞的质膜上也观察到强烈的反应。这些数据表明，成骨细胞和骨细胞中的LGP 107可能在细胞识别和/或细胞粘附中起重要作用，并且LGP 107可能参与类骨质的成骨细胞降解以及骨表面的暴露。3.通过体外免疫产生抗破骨细胞的单克隆抗体：本文对大鼠骨髓培养中形成的破骨样多核细胞（MNC_S）产生的单克隆抗体进行了免疫化学和生物化学研究。进行体外免疫后，获得单克隆抗体HOK 1。该抗体与基质细胞反应弱，与MNC_S及其在培养皿上的迁移痕迹反应强。用纯化的大鼠骨桥蛋白进行蛋白质印迹，证实HOK 1识别的抗原为骨桥蛋白。在MNC_S培养形成的骨吸收陷窝中进一步观察到HOK 1免疫反应阳性。目前的数据表明，骨桥蛋白是优先存在于吸收陷窝在再吸收钙化基质和破骨细胞可能会捕获这种蛋白质在其细胞表面。少

项目成果

Akifumi Akamine: "Expression and Localization of a Major Lysosomal Membrane Sialoglycoprotein(LGP107)in Plasma Membranes of Rat Osteoblasts and Osteocytes." Archives of Histology and Cytology. 56. 525-532 (1993)

Akifumi Akamine：“大鼠成骨细胞和骨细胞质膜中主要溶酶体膜唾液酸糖蛋白 (LGP107) 的表达和定位。”

Akifumi Akamine: "Increased Synthesis and Specific Localization of a Major Lysosomal Membrane Sialoglycoprotein(LGP107)at the Ruffled Border Membrane of Active Osteoclasts." Histochemistry. 100. 101-108 (1993)

Akifumi Akamine：“主要溶酶体膜唾液酸糖蛋白 (LGP107) 在活性破骨细胞褶皱边界膜上的合成和特异性定位增加。”

Akifumi Akamine, Takayuki Tsukuba, Ryusei Kimura, Katsumasa Maeda, Yoshitaka Tanaka, Keitaro Kato, and Kenji Yamamoto: "Expression and Localization of a Major Lysosomal Membrane Sialoglycoprotein (LGP107) in Plasma Membranes of Rat Osteoblasts and Osteocy

Akifumi Akamine、Takayuki Tsukuba、Ryusei Kimura、Katsumasa Maeda、Yoshitaka Tanaka、Keitaro Kato 和 Kenji Yamamoto：“大鼠成骨细胞和骨细胞质膜中主要溶酶体膜唾液酸糖蛋白 (LGP107) 的表达和定位

Akifumi Akamine: "Increased synthesis and specific localization of a major lysosomal membrane sialoglycoprotein(LGP107)at the ruffled border membrane of active osteoclasts" Histochemistry. 100. 101-108 (1993)

Akifumi Akamine：“主要溶酶体膜唾液酸糖蛋白 (LGP107) 在活性破骨细胞的褶皱边界膜上的合成和特异性定位增加”组织化学。

Akifumi Akamine, Takayuki Tsukuba, Ryusei Kimura, Katsumasa Maeda, Yoshitaka Tanaka, Keitaro Kato, and Kenji Yamamoto: "Increased Synthesis and Specific Localization of a Major Lysosomal Membrane Sialoglycoprotein (LGP107) at the Ruffled Border Membrane o

Akifumi Akamine、Takayuki Tsukuba、Ryusei Kimura、Katsumasa Maeda、Yoshitaka Tanaka、Keitaro Kato 和 Kenji Yamamoto：“增加主要溶酶体膜唾液酸糖蛋白 (LGP107) 在褶皱边界膜上的合成和特异性定位