Analysis of Genes Induced by Bone Morphogenetic Protein (BMP) in the Early Stage of Response

骨形态发生蛋白（BMP）早期反应诱导基因分析

• 批准号: 06807156
• 负责人: ASAHINA Izumi
• 金额: $ 1.28万
• 依托单位: Tokyo Medical and Dental 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: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807156/
• 关键词: BMP; OP-1; bone induction; gene; 骨形成因子; 骨芽細胞; 分化

To reveal the mechanism of action of Bone Morphogenetic Protein (BMP), we have examined the genes induced by BMP in the early stage of the response, which may control the action of BMP,using the differential display method in this study.We used Osteogenic Protein-1 (OP-1, BMP-7) as BMP,because highly purified recombinant OP-1 was available from Creative BioMolecule Inc. Examination of the effect of OP-1 on several cell lines revealed that OP-1 enhances alkaline phosphatase activity, one of the maker of osteoblastic characteristics, in the culture of osteoblastic MC3T3-E1 cells from mouse calvaria and stromal ST-2 cells from mouse bone marrow. After the treatment of OP-1 to these cells for 24hours, total RNA was extracted with AGPC method and reverse transcription was performed. According to the instruction of the kit for differential display method, polymerase chain reaction (PCR) on the resulted cDNA was performed with 20 sets of primer combination under the presence of 35S-dATP.The amplified cDNAs were electophoretically separated on a 6% sequence gel. Analysis of bands on the auto radiogram revealed that 66 cDNA fragments from MC3T3-E1 and 11 cDNA fragments from ST-2 were expressed differentially with or without the treatment of OP-1. cDNAs from MC3T3-E1 were subcloned and sequences were analyzed. There were a few clones which have homology with ubiquitin, SP-1, etc.but most of them were not identified clearly because the acquired sequences were only 200 to 300 base in 3'end region. Northern analysis was performed using these cDNA clones as a probe, but expression of most of them did not have any difference between with and without the treatment of OP-1. Therefore, we assumed that many cDNA clones obtained by differential display methods were psudepositive. However, we got two cDNA clones which show apparent difference on Northern analysis from ST-2. Sequencing analysis is on procedure.

为了揭示骨形态发生蛋白（Bone Morphogenetic Protein， BMP）的作用机制，本研究采用差异显示方法，检测了骨形态发生蛋白在反应早期诱导的可能控制BMP作用的基因。我们使用成骨蛋白-1 （OP-1, BMP-7）作为BMP，因为高纯度的重组蛋白OP-1可以从Creative biomolule公司获得。OP-1对几种细胞系的影响表明，在培养小鼠颅骨MC3T3-E1成骨细胞和小鼠骨髓基质ST-2细胞时，OP-1增强碱性磷酸酶活性，碱性磷酸酶是成骨特性的形成因子之一。OP-1作用于这些细胞24h后，用AGPC法提取总RNA并进行反转录。根据差异展示法试剂盒的说明，在35S-dATP存在下，用20组引物组合对得到的cDNA进行聚合酶链反应（PCR）。扩增的cdna在6%的序列凝胶上电泳分离。自动x线图条带分析显示，在OP-1处理或未处理的情况下，MC3T3-E1的66个cDNA片段和ST-2的11个cDNA片段的表达存在差异。对MC3T3-E1的cdna进行亚克隆和序列分析。有少数克隆与泛素、SP-1等具有同源性，但由于获得的序列仅在3′端区200 ~ 300个碱基，多数克隆未被明确鉴定。以这些cDNA克隆为探针进行了Northern分析，结果表明，在OP-1处理和未处理的情况下，大多数cDNA克隆的表达没有差异。因此，我们假设通过差异显示方法获得的许多cDNA克隆是假阳性的。然而，我们得到了两个cDNA克隆，它们与ST-2在Northern分析上有明显的差异。测序分析正在进行中。

项目成果

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Y.Maruoka, S.Oida, T.Iimura, K.Takeda, I.Asahina, S.Enomoto, and S.Sasaki: "Production of Functional Human Bone Morphogenetic Protein-2 Using a Baculovirus/ SF-9 Insect Cell System" Biochem Mol.Biol.Int.Vol 35 (5). 657-963 (1995)

Y.Maruoka、S.Oida、T.Iimura、K.Takeda、I.Asahina、S.Enomoto 和 S.Sasaki：“使用杆状病毒/SF-9 昆虫细胞系统生产功能性人骨形态发生蛋白-2”

Iimura, T., Oida, S., Takeda, K., Maruoka, Y.and Sasaki, S: "Changes in Homeobox-containing gene expression during ectopic bone formation induced by bone morphogenetic protein" Biochem.Biophys.Res.Commun. 201. 980-987 (1994)

Iimura, T.、Oida, S.、Takeda, K.、Maruoka, Y. 和 Sasaki, S：“骨形态发生蛋白诱导异位骨形成过程中含有同源盒的基因表达的变化”Biochem.Biophys.Res.Commun。