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

开发调节骨吸收药物的可靠筛选系统：破骨细胞形成和功能的体外测定系统。

基本信息

批准号：
05557082
负责人：
SUDA Tatsuo
金额：
$ 6.59万
依托单位：
Showa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

项目摘要

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

利用小鼠成骨细胞和骨髓细胞的共培养系统，我们建立了可靠的检测破骨细胞发育和功能的检测系统。在破骨细胞发育的测定系统中，在有丝分裂后破骨细胞前体分化成破骨细胞的过程中，检查了破骨细胞表型表达的时间变化。在本研究中，还使用这些测定系统检查了骨吸收抑制剂（如降钙素和双膦酸盐）在骨吸收中的作用机制。(1)分化为破骨细胞的有丝分裂后前体细胞的特征使用小鼠成骨细胞和骨髓细胞的共培养系统检测分化为破骨细胞的破骨细胞前体细胞的特征。有丝分裂后破骨细胞前体细胞是表达巨噬细胞相关表型如非特异性酯酶（NSE）、Mac-1和Mac-2的单核细胞。一些与巨噬细胞相关的表型 ...更多信息破骨细胞前体细胞在向破骨细胞分化的过程中PES迅速消失。(2)破骨细胞功能检测体系的建立我们以前建立了一种从小鼠成骨细胞和骨髓细胞的共培养物中制备大量功能活性破骨细胞的方法。为了研究骨吸收调节因子对破骨细胞功能的影响，我们已经开发了使用体外形成的破骨细胞进行小凹形成和肌动蛋白环形成的测定系统。我们已经证明破骨细胞的功能是由几种信号通路调节的，这些通路由环AMP依赖性蛋白激酶、酪氨酸激酶、磷脂酰肌醇-3激酶和小GTP结合蛋白rho介导。降钙素和二膦酸盐对破骨细胞骨吸收的影响降钙素和二膦酸盐对骨吸收的抑制作用的机制在上述建立的测定系统中进行了研究。降钙素和双磷酸盐抑制窝的破骨细胞放置在牙本质切片的活动。只有极性破骨细胞有皱褶的边界纳入双磷酸盐，抑制酪氨酸磷酸酶和破坏肌动蛋白环的破骨细胞。降钙素也诱导破骨细胞形态学的显着变化，包括破坏肌动蛋白环。在极化和非极化破骨细胞中均观察到这种效应。因此，降钙素和双磷酸盐抑制破骨细胞的功能，但它们的作用机制是完全不同的。少