Role of the osteoclast and osteoblast during the bone resorption

本课题的目的是研究破骨细胞和成骨细胞在骨吸收过程中的各自功能。我们应用体外培养系统来研究这些功能。本课题的研究结果如下：(1)用兔抗大鼠胶原酶抗体用间接免疫过氧化物酶技术检测了所有受试成骨细胞系(5个克隆大鼠成骨细胞系和1个大鼠骨肉瘤细胞系UMR-106)的成骨细胞胶原酶功能。B)组织纤溶酶原激活物是一种潜在的胶原酶刺激物，免疫组织化学方法检测到所有成骨细胞系中均有组织纤溶酶原激活物的表达。C)胶原酶活性测定采用AS试剂盒[胶原盒]。该试剂盒包含荧光标记的胶原蛋白作为胶原酶的底物。我们发现成骨细胞系的胶原酶活性在基础水平和…之间没有明显的增加甲状旁腺素或1，25(OH)_2D_3对成骨细胞胶原酶活性的刺激作用更强。这些结果表明，检测成骨细胞胶原酶活性时，应建立更灵敏的检测系统，包括[14C]标记的胶原酶。D)建立了新的骨降解测定系统，将成骨细胞培养在含或不含1，25(OH)_2D_3的~(13)H-Pro标记的颅骨上，培养5天后测定培养液和骨中~(13)H的含量。结果：1，25(OH)_2D_3刺激成骨细胞对胶原基质的降解。a)用兔抗大鼠胶原酶抗体间接免疫过氧化物酶技术检测所有受试成骨细胞系(5株克隆大鼠成骨细胞系和一株大鼠骨肉瘤细胞系UMR-106)均可检测到胶原酶。B)组织纤溶酶原激活物是一种潜在的胶原酶刺激物，免疫组织化学方法检测到所有成骨细胞系中均有组织纤溶酶原激活物的表达。C)胶原酶活性测定采用AS试剂盒[胶原盒]。该试剂盒包含荧光标记的胶原蛋白作为胶原酶的底物。在PTH或1，25(OH)_2D_3刺激条件下，成骨细胞株的胶原酶活性在基础水平和刺激条件下均未见明显升高。这些结果提示，可以测试更灵敏的检测系统，包括[^&lt；14&gt；C]标记的胶原酶，用于检测成骨细胞中的胶原酶活性。D)建立了新的骨降解率测定系统，将成骨细胞培养在含或不含1，25(OH)_2D_3的[~3H]-Pro标记的颅骨上，培养5天后测定培养液和骨中[~3H]的含量。结果表明，1，25(OH)_2D_3促进了成骨细胞对胶原基质的降解。(2)破骨细胞形成的研究)建立了小鼠骨髓细胞体外破骨细胞形成的检测体系。2)利用该系统，我们发现1，25(OH)_2D_3、PTH、IL-1、PGE_2促进破骨细胞的形成，而降钙素和干扰素-γ则抑制破骨细胞的形成。C)我们证明了小鼠脾细胞具有发育破骨细胞的潜力。在这个过程中，脾细胞需要成骨细胞、1，25(OH)_2D_3和地塞米松的存在。较少

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