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Search for key molecules in bone and cartilage differentiation

寻找骨和软骨分化的关键分子

基本信息

批准号：
13557153
负责人：
NIFUJI Akira
金额：
$ 7.68万
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 Scientific Research (B)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-13557153/
关键词：
Differentiation Bone cloning Cartilage BMP

项目摘要

Various type of signaling molecules and transcription factors are involved in bone and cartilage differentiation. Studies based on loss of function indicate that Runx 2, Osterix, and Sox 9 are indispensable for development of such hard tissues. Although those molecules are critical for skeletetogenesis, there still would be other uncharacterized molecules that govern bone and cartilage differentiation. To search for such molecules, we tried to establish a functional screening assay system specifically utilized for cartilage differentiation. We used type Xl collagen promoter as a monitor to differentiate into chondrocyte, since endogenous type Xl collagen expression is induced following the cartilage differentiation. We transfected collagen promoter plus beta-galactosidase construct (pXlcol-Z) into a teratocarcinoma derived fibroblastic cell line Fl 2. Using antibiotics we isolated stably expressed transformants. Among them, we selected one transformant, which barely expressed LacZ at steady state level but is induced to express upon treatment with BMP. Since it is known that BMP induced chondrogenic differentiation in vitro, BMP responsive promoter may reflect at least one aspect of chondrogenic differentiation. We named such transformants as pR5-1 cells. We next analyzed Lac~ Z expression by FACS. BMP treated pR5-l cells showed higher amount of LacZ positive cells than untreated cells, showing that FACS can be useful to collect cells in which the promoter is activated. We then prepared chondrocyte specific cDNAs. We used teratocarcinoma derived mesodermal cell line Cl as a source of RNA. We prepared cDNA libraries based on retrovirus vector plasmid and transfected them into a packaging cell line. Then we collected supernatant of those cells. We infected retrovirus cDNA library into pR5-1 cells. We, then, collected highly LacZ positive pR5-1 cells. We are now under way to isolate and characterize integrated genes.

各种类型的信号分子和转录因子参与骨和软骨分化。基于功能丧失的研究表明，Runx 2、Osterix和Sox 9对于这些硬组织的发育是必不可少的。尽管这些分子对骨骼发生至关重要，但仍有其他未表征的分子控制骨和软骨分化。为了寻找这样的分子，我们试图建立一个专门用于软骨分化的功能筛选分析系统。我们使用XI型胶原启动子作为分化成软骨细胞的监测器，因为软骨分化后诱导内源性XI型胶原表达。我们将胶原启动子加β-半乳糖苷酶构建体（pXlcol-Z）转染到畸胎癌衍生的成纤维细胞系F12中。使用抗生素，我们分离稳定表达的转化子。其中，我们选择了一种在稳态水平几乎不表达LacZ但在用BMP处理后被诱导表达的细胞。由于已知BMP在体外诱导软骨形成分化，因此BMP响应性启动子可以反映软骨形成分化的至少一个方面。我们将这样的转化体命名为pR 5 -1细胞。我们接下来通过FACS分析Lac~ Z表达。BMP处理的pR 5 - 1细胞显示比未处理的细胞更高量的LacZ阳性细胞，表明FACS可用于收集其中启动子被激活的细胞。然后我们制备了软骨细胞特异性cDNA。我们使用畸胎瘤衍生的中胚层细胞系Cl作为RNA的来源。我们制备了基于逆转录病毒载体质粒的cDNA文库，并将其转染到包装细胞系中。然后我们收集这些细胞的上清液。我们将逆转录病毒cDNA文库感染到pR 5 -1细胞中。然后，我们收集了高度LacZ阳性的pR 5 -1细胞。我们现在正在分离和鉴定整合基因。