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Dinamic and functional analysis of osteoclast activity using video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy

使用视频增强对比微分干涉对比 (VEC-DIC) 显微镜对破骨细胞活性进行动态和功能分析

基本信息

批准号：
17591561
负责人：
HOSHINO Hironobu
金额：
$ 2.3万
依托单位：
Hamamatsu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591561/
关键词：
osteoclast video microscopy pbase contrast microscope calcitonin bisphosphonate

项目摘要

Quantitative evaluation of the ability of bone resorption activity in live osteoclast-like cells (OCLs) has not reported yet. To analyze the ability of bone resorption in the same alive cells for long periods, we observed OCLs maintaining normal culture condition, and recorded by video: microscopy. The coverslips were placed in the incubator equipped inverted microscope (Biostation IM, Nikon, Japan), and OCLs were observed with phase contrast microscope. We observed the morphological change of OCLs and measured calcium phosphate free area made by OCLs affected by elcatonin using incubator facilitated video-enhanced microscopy. OCLs, which were obtained from co-culture of ddy-mouse osteoblastic cells and bone marrow cells, were cultured on calcium phosphate (CP)-coated quartz coverslips to observe sequential morphological change and to measure the increase of CP-free area. The CP-free area increase constantly in the control group, while that didn't increase after the addition of agent in the elcatonin group. In this study we could evaluate the resorbed areas increased on the same CP-coated coverslips, even with the migrated osteoclast. Furthermore, we observed the morphological change of OCLs after elcatonin addition. The nuclei were observed above the CP-free area and were gathered each other before chemical addition. After addition of elcatonin, however, the vacuoles spread from CP-free area, and the nuclei were dispersed. These results suggest that elcatonin could not only weaken the bone resorption ability but also change the polarization of nuclei. This study showed that analysis of the resorbed areas under the osteoclast-like cell body using this method enables the quantitative evaluation of the bone resorption activity to the several therapeutic agents like bisphosphonates in vitro.

定量评价活的破骨细胞样细胞（OCL）的骨吸收活性的能力还没有报道。为了分析同一活细胞长期存活的骨吸收能力，我们观察了OCL在正常培养条件下的生长情况，并通过视频显微镜进行记录。将盖玻片置于配备有倒置显微镜（Biotation IM，Nikon，Japan）的培养箱中，并用相差显微镜观察OCL。采用培养箱辅助视频增强显微镜观察依卡宁作用下OCL的形态学变化，并测量OCL形成的游离钙磷面积。将共培养的成骨细胞和骨髓细胞培养于磷酸钙（CP）包被的石英盖玻片上，观察OCL的形态学变化，并测量无CP面积的增加。对照组无CP区不断增加，而依卡宁组加药后无CP区增加。在本研究中，我们可以评价相同CP涂层盖玻片上增加的吸收面积，即使是迁移的破骨细胞。此外，我们还观察了加入依降钙素后OCL的形态学变化。在无CP区域上方观察到核，并且在化学品添加之前彼此聚集。加入依降钙素后，空泡从无CP区扩散，细胞核分散。结果提示，依卡宁不仅能减弱骨吸收能力，而且能改变细胞核的极化。该研究表明，使用该方法分析破骨细胞样细胞体下的吸收区域能够定量评价体外对几种治疗剂如双膦酸盐的骨吸收活性。