Akatsu T, Tamura T, Takahashi N, Udagawa N, Tanaka S, Sasaki T, Yamaguchi A, Nagata N, Suda T
Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan.
J Bone Miner Res. 1992 Nov;7(11):1297-306. doi: 10.1002/jbmr.5650071109.
We have reported that numerous tartrate-resistant acid phosphatase-positive osteoclast-like multinucleated cells (TRAP+ MNCs) are formed when mouse osteoblastic cells and spleen cells are cocultured in the presence of 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3] (Endocrinology 123:2600, 1988). In this study, we prepared a TRAP+ MNC population using a modified coculture system and examined its osteoclastic properties. TRAP+ MNCs were formed in cocultures of mouse osteoblastic cells and marrow cells on 10 cm collagen gel-coated dishes. The TRAP+ MNC population was prepared by treating the dishes with 0.2% bacterial collagenase followed by density gradient centrifugation. The yield of TRAP+ MNCs was 20,000-40,000 cells per dish, much higher than that of osteoclasts (OCLs) isolated from neonatal rat bones (approximately 1000 cells per head). The purity of TRAP+ MNCs was 5.6 +/- 0.6% in cell number and about 30% in the number of nuclei. The recovery of TRAP+ MNCs after density gradient centrifugation was 30-40%. Acid production by MNCs was demonstrated by vital staining with acridine orange. Numerous resorption pits were formed when the MNC population was cultured for 48 h on bone slices. Autoradiography using [125I]salmon calcitonin (CT) showed abundant CT binding in most TRAP+ MNCs. Saturation analysis of [125I]salmon CT indicated a dissociation constant Kd for TRAP+ MNCs of 8.9 +/- 0.7 x 10(-10) M and 16.5 +/- 1.5 x 10(6) binding sites per cell. These results were similar to the Kd value (3.5 x 10(-10) M) and the number of binding sites (3.3 x 10(6) per cell) in isolated rat OCLs. Displacement curves for [125I]salmon CT with unlabeled salmon and human CT were similar in MNC and OCL preparations. Salmon and human CT increased cAMP production (maximal response: slmon CT at 10(-10) M, human CT at 10(-8) M; ED50s: salmon CT, 2.2 x 10(-11) M, human CT, 1.3 x 10(-9) M) in the MNC preparation. These results indicate that a large number of mouse TRAP+ MNCs possessing OCL characteristics can be easily prepared from in vitro cultures. This procedure will facilitate examination of mammalian OCL functions.
我们曾报道，当小鼠成骨细胞与脾细胞在1α,25 - 二羟基维生素D3 [1α,25-(OH)2D3]存在的情况下共培养时，会形成大量抗酒石酸酸性磷酸酶阳性的破骨细胞样多核细胞（TRAP + MNCs）（《内分泌学》123:2600, 1988）。在本研究中，我们使用改良的共培养系统制备了TRAP + MNC群体，并检测了其破骨细胞特性。在10厘米胶原凝胶包被的培养皿上，小鼠成骨细胞与骨髓细胞共培养形成了TRAP + MNCs。通过用0.2%细菌胶原酶处理培养皿，然后进行密度梯度离心来制备TRAP + MNC群体。每个培养皿中TRAP + MNCs的产量为20,000 - 40,000个细胞，远高于从新生大鼠骨骼中分离的破骨细胞（OCLs）（每头约1000个细胞）。TRAP + MNCs在细胞数量上的纯度为5.6±0.6%，在细胞核数量上约为30%。密度梯度离心后TRAP + MNCs的回收率为30 - 40%。通过吖啶橙活体染色证明了MNCs产生酸。当MNC群体在骨切片上培养48小时时，形成了大量吸收陷窝。使用[125I]鲑鱼降钙素（CT）的放射自显影显示大多数TRAP + MNCs中有丰富的CT结合。[125I]鲑鱼CT的饱和分析表明，TRAP + MNCs的解离常数Kd为8.9±0.7×10(-10) M，每个细胞有16.5±1.5×10(6)个结合位点。这些结果与分离的大鼠OCLs中的Kd值（3.5×10(-10) M）和结合位点数（每个细胞3.3×10(6)个）相似。在MNC和OCL制剂中，[125I]鲑鱼CT与未标记的鲑鱼和人CT的置换曲线相似。鲑鱼和人CT增加了MNC制剂中的cAMP产生（最大反应：鲑鱼CT在10(-10) M，人CT在10(-8) M；半数有效剂量：鲑鱼CT为2.2×10(-11) M，人CT为1.3×10(-9) M）。这些结果表明，可以很容易地从体外培养物中制备出大量具有OCL特性的小鼠TRAP + MNCs。该方法将有助于对哺乳动物OCL功能的研究。
