Role of the osteoclast and osteoblast during the bone resorption

本项目旨在探讨破骨细胞和成骨细胞在骨吸收过程中的各项功能。我们利用体外培养系统来研究其功能。本项目的研究结果如下：(1)成骨细胞的功能a)采用间接免疫过氧化物酶技术，用兔抗大鼠胶原酶抗体检测所有成骨细胞（5株自行建立的克隆大鼠成骨细胞和1株大鼠骨肉瘤细胞系UMR-106）的免疫反应性胶原酶。b)组织纤溶酶原激活剂是一种潜伏型胶原酶的刺激剂，在所有的成骨细胞系中也检测到免疫组织化学技术。c)使用测定试剂盒[collagenokit]测定胶原酶活性。本试剂盒含有荧光共轭胶原蛋白作为胶原酶的底物。我们发现，在PTH或1,25(OH)_2D_3的刺激条件下，成骨细胞的胶原酶活性在基础水平和基础水平之间没有显著增加。这些结果建议测试更敏感的检测系统，包括[14C]标记的胶原蛋白用于检测成骨细胞中的胶原酶活性。d)我们开发了测量骨降解的新检测系统；将成骨细胞培养在13H]-脯氨酸标记的颅骨上，添加或不添加1,25(OH)_2D_3，培养5天后测定培养基和骨中的13H)水平。结果表明，1,25(OH)_2D_3刺激了成骨细胞对胶原基质的降解。a)采用间接免疫过氧化物酶技术，利用兔抗大鼠胶原酶抗体，在所有的成骨细胞系（5株自行建立的克隆大鼠成骨细胞系和1株大鼠骨肉瘤细胞系UMR-106）中检测到免疫反应性胶原酶。b)组织纤溶酶原激活剂是一种潜伏型胶原酶的刺激剂，在所有的成骨细胞系中也检测到免疫组织化学技术。c)使用测定试剂盒[collagenokit]测定胶原酶活性。本试剂盒含有荧光共轭胶原蛋白作为胶原酶的底物。我们发现，在基础水平和PTH或125 (OH)_2D_3刺激条件下，成骨细胞的胶原酶活性没有显著增加。这些结果建议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。d)我们开发了测量骨降解的新检测系统；将成骨细胞培养在[3H]-脯氨酸标记的颅骨上，添加或不添加1,25(OH)_2D_3，培养5天后测定培养基和骨中的[3H]水平。结果表明，1,25(OH)_2D_3刺激了成骨细胞对胶原基质的降解。(2)破骨细胞形成的研究(a)我们建立了小鼠骨髓细胞破骨细胞形成的体外实验系统。b)利用该系统，我们发现1,25(OH)_2D_3、PTH、IL-1、PGE_2刺激破骨细胞的形成，而降钙素和干扰素- γ抑制破骨细胞的形成。c)我们证明了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

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