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的方法，检测了BMP在反应早期诱导的可能调控BMP作用的基因。由于Creative Biolecule Inc.提供了高纯度的重组OP-1，我们选择了成骨蛋白-1(OP-1，BMP-7)作为BMP。OP-1对几种细胞系的作用检测表明，OP-1在小鼠颅骨成骨MC3T3-E1细胞和小鼠骨髓基质ST-2细胞培养中都能增强碱性磷酸酶的活性。OP-1作用于这些细胞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要求，在35S-dATP存在下，用20组引物组合进行聚合酶链式反应(PCR)，扩增出的cDNA在6%的序列凝胶上进行电泳性分离。放射自显影条带分析显示，经OP-1处理前后，MC3T3-E1和ST-2的66个基因片段和11个基因片段有差异表达。亚克隆了MC3T3-E1的基因，并进行了序列分析。有少数克隆与泛素、SP-1等有同源性，但由于获得的序列在3‘端只有200~300个碱基，大部分克隆没有得到明确的鉴定。以这些克隆为探针进行Northern分析，但其中大部分在OP-1处理前后的表达没有任何差异。因此，我们假设用差异显示方法获得的许多克隆都是假阳性的。但在Northern分析中，我们得到了两个与ST-2有明显差异的克隆。测序分析按程序进行。

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.