Takahashi N, Akatsu T, Sasaki T, Nicholson G C, Moseley J M, Martin T J, Suda T
Department of Biochemistry and Oral Anatomy, School of Dentistry, Showa University, Tokyo, Japan.
Endocrinology. 1988 Sep;123(3):1504-10. doi: 10.1210/endo-123-3-1504.
We have developed a mouse marrow culture system, in which multinucleated cells (MNCs) are formed within 6-8 days. These MNCs showed several characteristics of osteoclasts, including tartrate-resistant acid phosphatase (TRACP) and the ability to resorb calcified dentine. 1 alpha, 25-Dihydroxyvitamin D3 [1 alpha, 25 (OH)2D3] stimulated the formation of TRACP-positive MNCs, and salmon calcitonin (CT) inhibited it. In this study, we examined whether the TRACP-positive MNCs formed from mouse marrow cells possess CT receptors, another typical characteristic of osteoclasts. Mouse marrow cells cultured for 8 days with 10 nM 1 alpha, 25(OH)2D3 and freshly isolated authentic mouse osteoclasts were incubated with [125I]-salmon CT in the presence or absence of excess amounts of unlabeled CT, stained for TRACP, and processed for autoradiography. The [125I]-CT exclusively bound to TRACP-positive mononuclear cells and MNCs formed in the 1 alpha, 25(OH)2D3-treated cultures and also to isolated mouse osteoclasts. Both [125I]-CT binding and TRACP activity were induced simultaneously on mononuclear cells on day 3 in the cultures treated with 1 alpha, 25(OH)2D3. CT markedly stimulated cAMP production in the 1 alpha,25(OH)2D3-treated cultures. The CT-dependent cAMP production increased in parallel with the increase in the number of TRACP-positive MNCs formed. Neither freshly isolated marrow cells nor cells cultured without 1 alpha, 25(OH)2D3 showed CT-induced cAMP accumulation. Furthermore, CT induced cytoplasmic contraction of both marrow-derived MNCs and isolated osteoclasts. These results clearly indicate that 1 alpha,25(OH)2D3 induces TRACP activity and CT receptors almost simultaneously in mouse marrow cultures, and the MNCs formed in vitro respond to CT as authentic osteoclasts do.
我们开发了一种小鼠骨髓培养系统，在该系统中，多核细胞（MNCs）可在6 - 8天内形成。这些MNCs表现出破骨细胞的若干特征，包括抗酒石酸酸性磷酸酶（TRACP）以及吸收钙化牙本质的能力。1α,25 - 二羟基维生素D3 [1α,25(OH)2D3]刺激TRACP阳性MNCs的形成，而鲑鱼降钙素（CT）则抑制其形成。在本研究中，我们检测了从小鼠骨髓细胞形成的TRACP阳性MNCs是否具有CT受体，这是破骨细胞的另一个典型特征。将用10 nM 1α,25(OH)2D3培养8天的小鼠骨髓细胞和新鲜分离的正宗小鼠破骨细胞，在存在或不存在过量未标记CT的情况下，与[125I] - 鲑鱼CT一起孵育，进行TRACP染色，并进行放射自显影处理。[125I] - CT仅与在1α,25(OH)2D3处理的培养物中形成的TRACP阳性单核细胞和MNCs结合，也与分离的小鼠破骨细胞结合。在用1α,25(OH)2D3处理的培养物中，第3天单核细胞上同时诱导了[125I] - CT结合和TRACP活性。CT显著刺激了1α,25(OH)2D3处理的培养物中cAMP的产生。CT依赖性cAMP产生的增加与形成的TRACP阳性MNCs数量的增加平行。新鲜分离的骨髓细胞或未用1α,25(OH)2D3培养的细胞均未显示CT诱导的cAMP积累。此外，CT诱导了骨髓来源的MNCs和分离的破骨细胞的细胞质收缩。这些结果清楚地表明，1α,25(OH)2D3在小鼠骨髓培养物中几乎同时诱导TRACP活性和CT受体，并且体外形成的MNCs对CT的反应与正宗破骨细胞相同。
