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

为了揭示骨形态发生蛋白（BMP）的作用机制，本研究采用差异显示技术，检测了BMP在反应早期诱导的可能控制BMP作用的基因，我们选用成骨蛋白-1（OP-1，BMP-7）作为BMP，因为高纯度的重组OP-1可从Creative BioMolecule公司获得。OP-1对几种细胞系的作用的检查显示，OP-1增强碱性磷酸酶活性，碱性磷酸酶活性是成骨细胞特征的标志之一，在来自小鼠颅骨的成骨细胞MC 3 T3-E1细胞和来自小鼠骨髓的基质ST-2细胞的培养物中。OP-1作用24小时后，用AGPC法提取总RNA，进行逆转录。根据差异显示试剂盒的说明书，用20对引物组合在35 S-dATP存在下对所得cDNA进行聚合酶链反应（PCR），扩增的cDNA在6%测序胶上电泳分离。对自动放射照片上的条带的分析显示，来自MC 3 T3-E1的66个cDNA片段和来自ST-2的11个cDNA片段在OP-1处理或未处理的情况下差异表达。亚克隆来自MC 3 T3-E1的cDNA并分析序列。其中有少数克隆与泛素、SP-1等有同源性，但大多数克隆的3 '端序列只有200 ~ 300个碱基，鉴定不清楚。使用这些cDNA克隆作为探针进行北方分析，但是它们中的大多数的表达在OP-1处理和未处理之间没有任何差异。因此，我们认为，许多cDNA克隆获得的差异显示方法是假阳性。然而，我们从ST-2中得到了两个cDNA克隆，它们在北方分析中显示出明显的差异。测序分析正在进行中。

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.