A method for rapid demineralization of teeth and bones.
A method for rapid demineralization of teeth and bones.
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DOI：
10.2174/1874210601004010223
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发表时间：
2010-12-15
期刊：
影响因子：
--
通讯作者：
Kulkarni AB
中科院分区：
文献类型：
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作者：
Cho A;Suzuki S;Hatakeyama J;Haruyama N;Kulkarni AB
牙齿和骨骼标本需要广泛的脱矿，以仔细分析细胞形态以及基因和蛋白质表达水平。LacZ基因编码半乳糖苷酶，常被用作研究基因结构功能、启动子组织特异性表达、细胞谱系和命运的报告基因。该报告基因通过在目的启动子的控制下表达LacZ基因，在分析空间和时间基因表达模式方面特别有用。为了分析LacZ活性以及其他基因及其蛋白产物在牙齿和骨骼中的表达，有必要在切割切片之前对标本进行完全脱矿。然而，强酸，如用于牙齿脱矿的甲酸，会破坏包括半乳糖苷酶在内的酶的活性。因此，目前大多数方案使用软酸，如0.1M乙二胺四乙酸(EDTA)来进行牙齿和骨骼标本的脱矿，这需要较长的处理时间才能完全脱矿。一种可以快速、温和地对牙齿和骨骼等硬组织标本进行脱钙的方法，这是节省时间和精力所必需的。在这里，我们报告了一种合适的方法，在42˚C的0.1M乙二胺四乙酸乙二醇盐中快速脱矿，而不会损失β-半乳糖苷酶的活性。
Tooth and bone specimen require extensive demineralization for careful analysis of cell morphology, as well as gene and protein expression levels. The LacZ gene, which encodes the ß-galactosidase enzyme, is often used as a reporter gene to study gene-structure function, tissue-specific expression by a promoter, cell lineage and fate. This reporter gene is particularly useful for analyzing the spatial and temporal gene expression pattern, by expressing the LacZ gene under the control of a promoter of interest. To analyze LacZ activity, and the expression of other genes and their protein products in teeth and bones, it is necessary to carry out a complete demineralization of the specimen before cutting sections. However, strong acids, such as formic acid used for tooth demineralization, destroy the activities of enzymes including those of ß-galactosidase. Therefore, most protocols currently use mild acids such as 0.1 M ethylene diamine tetra-acetic acid (EDTA) for demineralization of tooth and bone specimen, which require a longer period of treatment for complete demineralization. A method by which hard tissue specimens such as teeth and bones can be rapidly, but gently, decalcified is necessary to save time and effort. Here, we report a suitable method for rapid demineralization of mouse teeth in 0.1M EDTA at 42˚C without any loss of ß-galactosidase activity.