Development of reliable screening systems for drugs which regulate bone resorption : In vitro assay systems for osteoclast formation and function.

利用小鼠成骨细胞和骨髓细胞的共培养体系，我们建立了检测破骨细胞发育和功能的可靠的检测系统。在破骨细胞发育的测试系统中，研究了有丝分裂后破骨细胞前体在分化为破骨细胞过程中表型表达的时间变化。本研究还探讨了降钙素和双膦酸类骨吸收抑制药在骨吸收中的作用机制。(1)有丝分裂后向破骨细胞分化的前体细胞的特性利用小鼠成骨细胞和骨髓细胞的共培养体系研究了破骨细胞前体细胞向破骨细胞分化的特性。有丝分裂后破骨细胞前体细胞是表达巨噬细胞相关表型的单核细胞，如非特异性酯酶(NSE)、Mac-1和Mac-2。一些与巨噬细胞相关的表型…破骨细胞前体在向破骨细胞分化过程中，有更多的PE迅速消失。(2)破骨细胞功能检测体系的建立我们已经建立了一种从小鼠成骨细胞和骨髓细胞共培养中大量制备具有功能活性的破骨细胞的方法。为了检测骨吸收调节因子对破骨细胞功能的影响，我们开发了利用体外形成的破骨细胞形成凹坑和肌动蛋白环的测定系统。我们发现Ostecast的功能受多种信号通路的调节，这些信号通路由环磷酸腺苷依赖的蛋白激酶、酪氨酸激酶、磷脂酰肌醇-3激酶和小的GTP结合蛋白Ro介导。降钙素和双磷酸盐对破骨细胞性骨吸收的影响。降钙素和双膦酸盐均可抑制牙本质片上破骨细胞的凹坑形成活性。只有具有褶皱边界的极化破骨细胞才会结合双膦酸盐，这会抑制酪氨酸磷酸酶并破坏破骨细胞的肌动蛋白环。降钙素还引起破骨细胞形态的显着变化，包括肌动蛋白环的破坏。在极化和非极化的破骨细胞中都观察到了这种效应。因此，降钙素和双膦酸盐都会抑制破骨细胞的功能，但它们的作用机制却截然不同。较少

Using a co-culture system of mouse osteoblastic cells and bone marrow cells, we have established reliable assay systems for examining osteoclast development and function. In a assay system for osteoclast development, chronological changes in phenotypic expression by postmitotic osteoclast precursors were examined during their differentiation into osteoclasts. The mechanism of action of bone resorption-inhibiting agents such as calcitonin and bisphosphonates in bone resorption was also examined in the present study using those assay systems.(1) Characteristics of postmitotic precursor cells that differentiate into osteoclastsCharacteristics of osteoclast precursors that differentiate into osteoclasts were examined using a co-culture system of mouse osteoblastic cells and bone marrow cells. Postmitotic osteoclast precursors were mononuclear cells which expressed macrophage-associated phenotypes such as nonspecific esterase (NSE), Mac-1 and Mac-2. Some of the macrophage-associated phenoty … More pes in osteoclast precursors disappeared rapidly during their differentiation into osteoclasts.(2) Establishment of assay systems for examining osteoclast functionWe have previously established a method for preparing a large number of functionally active osteoclasts from cocultures of mouse osteoblastic cells and bone marrow cells. To examine effects of bone resorption-regulatory factors on osteoclast function, we have developed assay systems for pit formation and actin ring formation using osteoclasts formed in vitro. We have shown that osteclast function is regulated by several signaling patheays mediated by cyclic AMP dependentprotein kinase, tyrosine kiases, phosphatidylinositol-3 kinase, and small GTP binding protein, rho.Effects of calcitonin and bisphosphonates on osteoclastic bone resorptionThe mechanism of inhibitory action of calcitonin and bisphosphonates on bone resorption was examined in established assay systems as described above. Both calcitonin and bisphosphonates inhibited pit forming activity of osteoclasts placed on dentine slices. Only polarized osteoclasts having ruffled borders incorporated bisphosphonates, which inhibited tyrosine phosphatases and disrupted actin rings of osteoclasts. Calcitonin also induced marked changes in osteoclast morphology including disruption of actin rings. This effect was observed in both polarized and non-polarized osteoclasts. Thus, both calcitonin and bisphosphonates suppress osteoclast function, but their mechanisms of action are quite different from each other. Less