Development of new assay systems for examining the relation between osteoblasts and osteoclasts, and identification of new factors controlling bone metabolism

本研究的目的是开发新的检测系统来检测成骨细胞和破骨细胞之间的关系，并确定控制骨代谢的新因素。为了解决这些问题，我们进行了以下实验：(1)分离和表征成骨细胞产生的成骨因子，(2)开发用于体外检测破骨细胞形成的新检测系统，(3)阐明成骨细胞在破骨细胞形成中的作用。我们之前报道成骨细胞MC3T3-E1产生分化诱导因子（DIF），促进小鼠髓系白血病细胞（M1）向巨噬细胞样细胞分化。从MC3T3-E1细胞的条件培养基中部分纯化DIF，并检测其在骨吸收中的生物活性。在Raisz试验系统中，DIF表现出明显的骨吸收活性，并在小鼠骨髓培养系统中刺激破骨细胞样细胞的形成。我们开发了一种小鼠骨髓培养系统，在体外检测破骨细胞的形成。1 α、25(OH)_2D_3、PTH、PGE_2、IL-1、TNFalpha、TGF等骨吸收刺激激素和因子能显著促进破骨细胞样多核细胞的形成，而降钙素和IFN<@2Y<@D2则能强烈抑制这些激素和因子的形成。为了研究成骨细胞在破骨细胞形成中的作用，我们建立了小鼠脾细胞和从胎鼠颅骨分离的成骨细胞的共培养系统。小鼠脾细胞与成骨细胞在1alpha， 25(OH)_2D_3的存在下共培养，8天内形成破骨细胞样MNCs。不含维生素的同样的共培养，或含维生素的脾细胞或成骨细胞的单独培养都不能产生破骨细胞样跨国公司。这些结果表明，成骨细胞是破骨细胞祖细胞向多核破骨细胞分化所必需的。少

The aim of the present study was to develop new assay systems for examining the relation between osteoblasts and osteoclasts and to identify new factors controlling bone metabolism. In order to address these problems, we performed following experiments: (1) isolation and characterization of osteotropic factors produced by osteoblasts, (2) development of new assay systems for examining osteoclast formation in vitro, and (3) clarification of the role of osteoblasts in osteoclast formation.1. We previously reported that osteoblastic MC3T3-E1 cells produced differentiation inducing factor (DIF) which promoted differentiation of mouse myeloid leukemia cells (M1) into macrophage-like cells. The DIF was partially purified from the conditioned meda of MC3T3-E1 cells and its biological activity in bone resorption was examined. The DIF exhibited a marked bone resorbing activity in a Raisz's assay system and it stimulated osteoclast-like cell formation in a mouse marrow culture system.2. We devel … More oped a mouse bone marrow culture system to examine osteoclast formation in vitro. Bone resorption-stimulating hormones and factors such as 1alpha,25(OH)_2D_3, PTH, PGE_2, IL-1, TNFalpha, and TGF markedly stimulated the formation of osteoclast-like multinucleated cells (MNCs), whereas calcitnin and IFN<@2Y<@D2 strongly inhibited the formation induced by those hormones and factors.3. In order to examine the role of osteoblasts in osteoclast formation, we developed a co-culture system of mouse spleen cells and osteoblastic cells freshly isolated from fetal mouse calvariae. When mouse spleen cells were co-cultured with osteoblastic cells in the presence of 1alpha, 25(OH)_2D_3, osteoclast-like MNCs were formed within 8 days. Neither the same co-culture without the vitamin nor separate cultures of either spleen cells or osteoblastic cells with the vitamin produced osteoclast-like MNCs. These results indicate that osteoblasts are required for differentiation of osteoclast progenitors into multinucleated osteoclasts. Less