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Establishment of a osteoblastic cell line derived from calvarial of rat embryo.

大鼠胚胎颅骨来源的成骨细胞系的建立。

基本信息

批准号：
04671117
负责人：
HISADA Yoh
金额：
$ 1.41万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1994
项目状态：
已结题

项目摘要

An osteoblastic cell line that retains the capacity to mineralize in vitro would be a very useful model system for the study of the mechanism of calcification.However a few lines have established including the origin of cancer cells.The present study aimed to establish the cell line from calvariae of 18-21-old rat embryo.Isolation of osteoblastic cells : (1) Single-cell suspensions from enzymatic digestions of the calvariae.(2) Outgrowth from the flagments of calvariae.Culture medium : alpha-MEM with 10% FBS AND 60mug/ml Kanamycine sulfate.The changes of media at every 2 days.Subculture at 7-10 days with confluence state, in the culture.Passage culture : Cells obtained by the populations to form opaque regions representing mineralized areas within the nodules.ALPase activity : Assay by the method with pNPP.Electoum Microscopy : Scanniry and transmission electron microscope observations.[Results]About 18 cell lines induced from both of isolated methods have established showing the nodul … More es with mineralization.These lines were stored in liguid nitrogen at-80ﾟC.Some cell lines continued to more 80th passage culture.Especially, these cell lines were studied to characterize the processes of differentiation of cells and calcification in vitro.ALP activity in each cell line showed very low at the beginning of culture, then increased after the confluence state of cells.These results strongly suggested that the cells were an osteogenic cell line.In the cells of 52-55 passage culture, the formation of mineralized tissue were observed from the 2-days to 28 weeks in the culture period as follows : Monolayr of osteoblast-like cells at 2-days.Two or more layrs of the cells at 7-days.Appearance of nodules and collagenouse matrix at 2-weeks.The nodules comprised a collagenouse matrix containing ovoid osteocyte-like cells and needle-shape crystals containing Ca^<2+> and Pi within 28 weeks.From these results, these cell lines are an established clonal osteoblastic cell line, and it should be a useful model system for study of the mechanism of biological calcification. Less

在体外保留矿化能力的成骨细胞系将是研究钙化机制的一个非常有用的模型系统。然而，已经建立了一些线索，包括癌细胞的起源。本研究旨在建立18-21岁大鼠胚胎颅骨细胞系。成骨细胞的分离：(1)从颅骨酶消化中提取单细胞悬液。(2)胼胝体鳞片的外生物。培养基：α - mem与10% FBS和60mug/ml硫酸卡那胺。每2天更换一次介质。继代培养7-10天，达到融合状态。传代培养：由群体获得的细胞在结节内形成代表矿化区域的不透明区域。ALPase活性：pNPP法测定。电子显微镜：扫描和透射电子显微镜观察。[结果]两种分离方法诱导的18株细胞株均显示出矿化度较高的结节。这些细胞系保存在液氮中，温度为80℃。部分细胞系继续培养到80代以上。我们特别研究了这些细胞系的细胞分化和体外钙化过程。各细胞系的ALP活性在培养开始时表现为极低，在细胞融合状态后逐渐升高。这些结果强烈提示该细胞为成骨细胞系。在培养52 ~ 55代的细胞中，从培养第2天到培养第28周观察到矿化组织的形成情况如下：第2天形成成骨样细胞单层。7天形成两层或多层细胞。2周时出现结节和胶原基质。28周内结节由含有卵形骨细胞样细胞的胶原基质和含有Ca^<2+>和Pi的针状晶体组成。由此可见，这些细胞系是已建立的克隆成骨细胞系，为研究生物钙化机制提供了一个有用的模型系统。少