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Molecular Biological and Immunohistochemical Study of Differentiation of the Osteoblast.

成骨细胞分化的分子生物学和免疫组织化学研究。

基本信息

批准号：
01570995
负责人：
IKEDA Tohru
金额：
$ 1.34万
依托单位：
Showa 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：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570995/
关键词：
Osteoblast Differentiation Gla protein In situ hybridization Bone tissue Cultured cell Molecular biology 遺伝子 in situ hybridization

项目摘要

In the studies supported by this grant, the following results were cleared.1. The expression of BGP and MGP genes in the rat osteoblastic cell lines, C11, C20, C23 and C26 was studied by the method of Northern blot hybridization. All the cell lines expressed MGP gene, but did not express BGP gene. After administration of l,25-dihydroxyvitamin D_3, the levels of MGP gene expression was greatly increased except for C26 cells, which are in more immature stage of osteoblastic differentiation than the other cell lines.2. We found that recombinant human bone morphogenetic protein-2 (BMP-2) stimulates osteoblastic maturation of C26 cells in the collaboration study with Genetics Institute Inc.3. The mouse and rat MGP cDNAs were cloned from the cDNA libraries constructed from mouse osteoblastic cell line MC3T3-El, and the cell line C20, respectively. As a result of the nucleotide sequencing, the cloned mouse MGP cDNAs were found that one of the five glutamic acid residues potentially modified to Gla not conserved.4. To study differentiation of osteoblast in the bone tissue, wo developed the new method for in situ hybridization of non-decalcified bone tissue. Using this method, we studied the expression of BGP and MGP genes in the femur and lumbar of adukt rats. BGP gene was strongly expressed in cuboidal osteoblasts along the mineralized bone traveculars. On the other hand. MGP gene was expressed strongly in hypertrophic chondrocytes. From these results, the expression of BGP and MGP genes in immature bone tissue before mineralization was thought to be different from the expression in mature bone tissue.

本次资助的研究取得了以下成果： 1．采用Northern blot杂交方法研究了大鼠成骨细胞系C11、C20、C23和C26中BGP和MGP基因的表达。所有细胞系均表达MGP基因，但不表达BGP基因。给予1,25-二羟基维生素D_3后，除C26细胞外，其他细胞系的MGP基因表达水平均显着升高，其成骨分化阶段较其他细胞系更为不成熟。 2．在与Genetics Institute Inc.的合作研究中，我们发现重组人骨形态发生蛋白2（BMP-2）可刺激C26细胞的成骨细胞成熟3。小鼠和大鼠MGP cDNA分别从小鼠成骨细胞系MC3T3-E1和细胞系C20构建的cDNA文库中克隆。核苷酸测序结果发现，克隆的小鼠MGP cDNAs中5个可能修饰为Gla的谷氨酸残基之一不保守。4．为了研究骨组织中成骨细胞的分化，我们开发了非脱钙骨组织原位杂交的新方法。利用该方法，我们研究了成年大鼠股骨和腰椎中BGP和MGP基因的表达。 BGP 基因在沿矿化骨小梁的立方成骨细胞中强烈表达。另一方面。 MGP基因在肥大软骨细胞中强烈表达。从这些结果来看，矿化前的未成熟骨组织中的BGP和MGP基因的表达被认为与成熟骨组织中的表达不同。