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Studies on Ca^<2+> mobilization in osteoblasts and in odontblasts in primary culture.

原代培养中成骨细胞和成牙本质细胞中Ca ^ 2 动员的研究。

基本信息

批准号：
62480375
负责人：
ISHIKAWA Ichijiro
金额：
$ 3.65万
依托单位：
Niigata University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

It is generally accepted that cytosolic free Ca^<2+> ([Ca^<2+>]i) is one of second messengers as is cAMP. We had investigated the effect of parathyroid hormone (PTH) on alkaline phosphatase (ALP) activity and cAMP production in the presence or absence of extracellular Ca^<2+> at various concentrations in rat dental pulp cells (DP cells), including odontblasts. To compare this responses of DP cells with that of bone-forming cells (BF cells, i.e. osteoblast-like cells) in primary culture, we examined the effects of various reagents on [Ca^<2+>]i in BF cells.In the first year, we established the isolation technique of BF cells from newborn (or neonatal) mouse calvaria using sequential enzyme digestion. Furthermore, we studied the basic technique of subculture with maintaining the differentiated functions of the cells. On the other hand, to master the method of [Ca^<2+>]i measurement using a Nikon microspectrofluorometry system, we first examined the effect of NaF on [Ca^<2+>]i in single L … More -929 mouse fibroblasts cultured on coverslips (the single cell method). F- induced a transient increase in [Ca^<2+>]i.In BF cells in premary culture, we demonstrated that [Ca^<2+>]i rapidly elevated in response to phosphatidic acid (PA). In primary cultures, however, the single cells method is unsuitable to [Ca^<2+>]i measurement, since it is impossible to avoid contamination of many cell types. such as fibroblasts. To resolve this problem, we tried to establish an osteoblast-like cell line.In the final year, we established a clonal osteoblast-like cell line (MOB 3-4) derived from neonatal mouse calvaria. In the cells, an increase in the culture density enhanced ALP activity and induced the response to PTH. The pattern of the NaF-increased [Ca^<2+>]i was similar to that of the PTH action in the cells in a dense culture. In addition, the cells display many osteoblastic characteristics including high bone-specific ALP activity, production of I collagen, and the responses to PTH, PGE2, and 1,25(OH)2D3 resulting in increased ALP activity and [Ca^<2+>]i.In conclusion, it is suggested that cytosolic free Ca^<2+> may modulate the regulation of ALP activity in a clonal osteoblast-like cell line, MOB 3-4, and that this modulation by Ca^<2+> may be applied to odontblasts. Less

胞浆游离Ca^2+（[Ca^2+]i）与cAMP一样是第二信使。我们研究了在不同浓度的细胞外Ca^2+存在或不存在的情况下，甲状旁腺激素（PTH）对大鼠牙髓细胞（DP细胞）（包括成牙本质细胞）碱性磷酸酶（ALP）活性和cAMP生成的影响。为了比较原代培养的DP细胞和成骨细胞（BF细胞，即成骨样细胞）的这种反应，我们检测了不同试剂对BF细胞[Ca^<2+>]i的影响。在此基础上，研究了在保持细胞分化功能的前提下进行传代培养的基本技术。另一方面，为了掌握使用Nikon显微荧光分光光度计系统测量[Ca ^<2 +>] i的方法，我们首先检查了NaF对单个L ...更多信息 -929在盖玻片上培养的小鼠成纤维细胞（单细胞方法）。F-可引起[Ca^<2+>]i的短暂升高。在原代培养的BF细胞中，我们证明[Ca^<2+>]i对磷脂酸（PA）的反应迅速升高。然而，在原代培养中，单细胞法不适合于[Ca^2+]i测量，因为它不可能避免许多细胞类型的污染。如成纤维细胞。为了解决这一问题，我们尝试建立成骨样细胞系，并在最后一年建立了一个新生小鼠颅骨成骨样细胞系（MOB 3-4）。在细胞中，培养密度的增加增强ALP活性并诱导对PTH的反应。NaF增加[Ca^<2+>]i的模式与高密度培养细胞中PTH作用的模式相似。此外，这些细胞显示出许多成骨细胞的特征，包括高骨特异性ALP活性，I型胶原的产生，以及对PTH、PGE 2和1，25（OH）2D 3的反应，导致ALP活性和[Ca^2+]i增加。总之，细胞溶质游离Ca^2+可能调节克隆成骨样细胞系MOB 3-4中ALP活性的调节，而且Ca^2+的这种调节作用可能也适用于成牙本质细胞。少