Role of the osteoclast and osteoblast during the bone resorption

破骨细胞和成骨细胞在骨吸收过程中的作用

• 批准号: 63480406
• 负责人: YOSHIKI Shusaku
• 金额: $ 4.03万
• 依托单位: Showa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480406/
• 关键词: osteoblast; osteoclast; bone resorption; collagenase; PTH; 1,25(OH)_2D_3; 副甲状腺ホルモン; コラゲナーゼ

The object of this Project is to investigate each function of the osteoclast and osteoblast during bone resorption process. We applied in vitro culture systems for studying the functions. The results obtained from this project are as follows.(1)Function o-f the osteoblast a)Immunoreactive-collagenase was detected in all of the osteoblastic cell lines tested (5 clonal rat osteoblastic cell lines established by ourselves and a rat osteosarcoma cell line, UMR-106) using a rabbit anti-rat collagenase antibody by an indirect immunoperoxidase technique. b)Tissue plasminogen activator, which is a stimulator of latent form-collagenase, was also detected in all of the osteoblastic cell lines tested by an immunohistochemical technique. c) Collagenase activity was measured using as assay kit [collagenokit]. This kit contains fluorescence- conjugated collagen as substrate for collagenase. We found no significant increase of collagenase activity in the osteoblastic cell lines between basal level an … More d stimulated conditions with PTH or 1,25(OH)_2D_3. These results suggested to test the more sensitive assay system, including [14C] labeled collagen 'for the detection of the collagenase activity in the osteoblastic cells. d) We developed new assay system for the measurement of bone degradation;osteoblastic cells were cultured on the 13H]-proline labeled calvaria with or without 1,25(OH)_2D_3, and the level of 13H) was determined in the medium and bone after 5 days culture. As a result, 1,25(OH)_2D_3 stimulated the degradation of collagenous matrix by the osteoblastic cells.a)Immunoreactive-collagenase was detected in all of the osteoblastic cell lines tested (5 clonal rat osteoblastic cell lines established by ourselves and a rat osteosarcoma cell line, UMR-106) using a rabbit anti-rat collagenase antibody by an indirect immunoperoxidase technique. b)Tissue plasminogen activator, which is a stimulator of latent form-collagenase, was also detected in all of the osteoblastic cell lines tested by an immunohistochemical technique. c) Collagenase activity was measured using as assay kit [collagenokit]. This kit contains fluorescence- conjugated collagen as substrate for collagenase. We found no significant increase of collagenase activity in the osteoblastic cell lines between basal level and stimulated conditions with PTH or 1,25(OH)_2D_3. These results suggested to test the more sensitive assay system, including [^<14>C] labeled collaben, for the detection of the collagenase activity in the osteoblastic cells. d) We developed new assay system for the measurement of bone degradation;osteoblastic cells were cultured on the [3H]-proline labeled calvaria with or without 1,25(OH)_2D_3, and the level of [3H] was determined in the medium and bone after 5 days culture. As a result, 1,25(OH)_2D_3 stimulated the degradation of collagenous matrix by the osteoblastic cells.(2)Study for the osteoclast formationa) We developed an in vitro assay system for the osteoclast formation from mouse bone marrow cells. b)Using this system, we found that 1,25(OH)_2D_3, PTH, IL-1, PGE_2 stimulate the osteoclast formation, and calcitonin and interferon-gamma inhibit its formation. c) We demonstrated that mouse spleen cells have a potential to develop the osteoclast. In this process, spleen cells required the presence of the osteoblastic cells, 1,25(OH)_2D_3 and dexamethasone. Less

本课题旨在研究破骨细胞和成骨细胞在骨吸收过程中各自的功能。我们应用体外培养系统来研究其功能。该项目取得的成果如下。（1）成骨细胞的功能a）用兔抗大鼠胶原酶抗体通过间接免疫过氧化物酶技术在所有测试的成骨细胞系（5个我们建立的克隆大鼠成骨细胞系和大鼠骨肉瘤细胞系UMR-106）中检测免疫反应性胶原酶。B）组织纤溶酶原激活物，其是潜伏型胶原酶的刺激物，也在通过免疫组织化学技术测试的所有成骨细胞系中检测到。c）使用测定试剂盒[collagenokit]测量胶原酶活性。该试剂盒含有荧光缀合的胶原蛋白作为胶原酶的底物。我们发现，在成骨细胞系中，胶原酶活性在基础水平和基础水平之间没有显著增加， ...更多信息 d用PTH或1，25（OH）_2D_3刺激条件。这些结果表明，测试更敏感的检测系统，包括[14 C]标记的胶原蛋白，用于检测成骨细胞中的胶原酶活性。（4）建立了一种新的骨降解检测系统，将成骨细胞培养在13 H]-脯氨酸标记的颅骨上，在1，25（OH）_2D_3存在或不存在的情况下，培养5天后测定培养液和骨中13 H）的含量。结果表明，1，25（OH）_2D_3能刺激成骨细胞降解胶原基质。a）用兔抗大鼠胶原酶抗体，采用间接免疫过氧化物酶技术，检测到5株大鼠成骨细胞系和1株大鼠骨肉瘤细胞系UMR-106中均存在免疫反应性胶原酶。B）组织纤溶酶原激活物，其是潜伏型胶原酶的刺激物，也在通过免疫组织化学技术测试的所有成骨细胞系中检测到。c）使用测定试剂盒[collagenokit]测量胶原酶活性。该试剂盒含有荧光缀合的胶原蛋白作为胶原酶的底物。我们发现在基础水平和PTH或1，25（OH）_2D_3刺激条件下，成骨细胞系中胶原酶活性没有显著增加。这些结果表明，测试更敏感的检测系统，包括[^<14>C]标记的胶原酶，用于检测成骨细胞中的胶原酶活性。（4）建立了一种新的骨降解检测系统，将成骨细胞培养在[~ 3 H]-脯氨酸标记的颅骨上，分别加入或不加入1，25（OH）_2D_3，培养5天后测定培养液和骨中的[~ 3 H]水平。结果表明，1，25（OH）_2D_3可促进成骨细胞降解胶原基质。（2）破骨细胞形成的研究a）建立了小鼠骨髓细胞破骨细胞形成的体外实验系统。B）1，25（OH）_2D_3、PTH、IL-1、PGE_2刺激破骨细胞的形成，降钙素、γ-干扰素抑制破骨细胞的形成。c）我们证明小鼠脾细胞具有产生破骨细胞的潜力。在此过程中，脾细胞需要成骨细胞、1，25（OH）_2D_3和地塞米松的存在。少

项目成果

Yamaguchi, A., Yoshiki, S.: "Diversity of phenotype and function in osteoblast." J. Clin. Sci. 25, 241-248, 1989.

Yamaguchi, A.，Yoshiki, S.：“成骨细胞表型和功能的多样性。”

Takahashi, N., Yamana, H., Yoshiki, S., Roodman, G.D., Mundy, G.R., Jones, S.J., Boyde, A., Suda, T.: "Osteoclast-like cell formation and its regulation by osteotropic hormones in mouse bone marrow" Endocrinology; 122, 1373-1382, 1988.

Takahashi, N.、Yamana, H.、Yoshiki, S.、Roodman, G.D.、Mundy, G.R.、Jones, S.J.、Boyde, A.、Suda, T.：“破骨细胞样细胞的形成及其对成骨激素的调节

山口朗,吉木周作: "骨のコラゲナ-ゼ" 骨代謝誌. 7. 50-57 (1989)

Akira Yamaguchi、Shusaku Yoshiki：“骨胶原酶”《骨代谢杂志》7. 50-57 (1989)。

Yamaguchi, A., Yoshiki, S.: "Bone collagenase." Jap. Soc. Bone Mineral Res. 7.50-57, 1989.

Yamaguchi, A.，Yoshiki, S.：“骨胶原酶。”

Yamaguchi, A., Kahn, A.J.: "Clonal osteogenic cell lines express myogenic and adipocytic developmental potential." submitted.

Yamaguchi, A., Kahn, A.J.：“克隆成骨细胞系表达肌源性和脂肪细胞发育潜力。”