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Establishment of the screening methods for bone-resorbing factors using in vitro osteoclast formation system.

体外破骨细胞形成系统筛选骨吸收因子方法的建立

基本信息

批准号：
01870078
负责人：
SUDA Tatsuo
金额：
$ 7.42万
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
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

We have developed in vitro systems to examine the effects of osteotropic factors on the sequential process of osteoclastic bone resorption ; 1) proliferation of osteoclast progenitors, 2) differentiation of osteoclast precursors into multinucleated osteoclasts, and 3) pit formation by functionally active osteoclasts.1. Evaluation of proliferation of osteoclast progenitorsWe have developed a two-step culture system to determine the effect of osteotropic factors on proliferation of osteoclast progenitors. Bone marrow cells were first cultured in semisolid methylcellulose in the presence of various CSFs. Marrow cells were then isolated and further co-cultured with osteoblastic cells in the presence of 1alpha, 25(OH)_2D_3. After co-culture for 7 days, the number of osteoclasts formed were scored. On the basis of the number of marrow cells co-cultured with osteoblastic cells and that of osteoclasts formed, we were able to estimate the number of osteoclast progenitors present in marrow cell … More fractions. Using this method, we found that M-CSF was the most potent growth factor in inducing the growth of osteoclast progenitors.2. Evaluation of differentiation of osteoclast precursorsWe have reported that osteoclasts are formed in mouse marrow cultures and in co-cultures of mouse osteoblastic cells and spleen cells. Using these cultures, we have established a screening system for examining the effects of osteotropic factors on osteoclast differentiation. Bone-resorbing factors such as 1alpha, 25(OH)_2D_3, PTH, PGE_2 and IL-1 similarly stimulated differentiation of osteoclast precursors into functionally active osteoclasts.3. Evaluation of bone-resorbing activity of osteoclastsOsteoclasts formed on plastic dishes were hardly released from the dish surface. In contrast, when co-cultures were performed on collagen gel-coated dishes then treated with collagenase, most of the cells were easily released from the dishes. The osteoclast population was enriched by density gradient centrifugation. Using an osteoclast-enriched population and dentine slices, we have developed a simple bone resorption assay system. When isolated osteoclasts were cultured on dentine slices, they formed resorption pits within 24 hr. The area of resorption pits was quantitatively measured with an image analyzer. Using this system, we found that calcitonin and bafiromycin A_1 (an inhibitor of vacuolar H^+-ATPase) strongly inhibited pit formation by isolated osteoclasts.These systems appear to be useful for examining the mechanism of action of osteotropic factors in osteoclastic bone resorption. Less

我们已经开发了体外系统来检测促骨因子对破骨细胞性骨吸收的一系列过程的影响：1)破骨细胞前体的增殖，2)破骨细胞前体向多核破骨细胞的分化，以及3)功能活跃的破骨细胞形成凹陷1。破骨细胞前体细胞增殖的评价我们建立了一种两步培养体系，以确定促骨因子对破骨细胞前体细胞增殖的影响。首先在含有不同CSF的半固体甲基纤维素中培养骨髓细胞。分离骨髓细胞，在1α，25(OH)2D3存在下与成骨细胞共培养，共培养7d后计算破骨细胞数。根据与成骨细胞共培养的骨髓细胞和形成的破骨细胞的数量，我们能够估计骨髓细胞…中存在的破骨细胞前体细胞的数量。更多的分数。结果：1.利用该方法，我们发现M-CSF是诱导破骨细胞前体细胞生长的最有效的生长因子。破骨细胞前体细胞分化的评价我们已经报道在小鼠骨髓培养以及小鼠成骨细胞和脾细胞的共同培养中形成破骨细胞。利用这些培养物，我们建立了一个筛选系统，用于检测促骨因子对破骨细胞分化的影响。骨吸收因子1α、25(OH)_2D_3、PTH、PGE_2和IL-1类似地刺激破骨细胞前体向功能活跃的破骨细胞分化。破骨细胞对骨吸收活性的评价塑料培养皿上形成的破骨细胞很难从培养皿表面释放出来。相反，在胶原胶涂层的培养皿上进行共培养，然后用胶原酶处理，大多数细胞很容易从培养皿中释放出来。密度梯度离心法丰富破骨细胞群。利用富含破骨细胞的群体和牙本质切片，我们开发了一种简单的骨吸收测定系统。当分离的破骨细胞在牙本质切片上培养时，它们在24小时内形成吸收坑。用图像分析仪定量测量吸收陷窝面积。利用该系统，我们发现降钙素和Bafiromycin A_1(空泡H~+-ATPase的抑制剂)对体外培养的破骨细胞的凹陷形成有强烈的抑制作用。这些系统可用于研究促骨因子在破骨细胞性骨吸收中的作用机制。较少