Strategy to assess the function of osteocyte restricted genes

描述(由申请人提供)： 骨细胞是从成骨细胞分化而来的终末分化细胞，成骨细胞被包裹在自己的基质中。骨细胞通过建立相互连接的树突小管网络与其他骨细胞和其他成骨细胞谱系细胞进行交流而生存。众所周知，骨组织具有改变其质量和结构的能力，以骨细胞为机械传感细胞来响应机械应变。识别参与这些过程的基因需要分离纯净的骨细胞群体，并与成骨细胞谱系中其他成熟阶段的细胞的基因表达谱进行比较。在这个项目中，我们建议评估一个分离的骨细胞群体的基因表达谱，并将其与成骨细胞在进入成骨细胞阶段时的基因表达进行比较。将利用膜内成骨过程中小鼠颅骨成骨细胞分化的明确模型。所有成熟阶段的细胞将通过对小鼠的顶骨进行连续的酶消化来分离，这些小鼠的顶骨含有牙本质基质蛋白1(DMP-1)启动子驱动的GFPtopaz。这个启动子已经被证明驱动GFP的表达到被包裹在基质中的细胞(前骨细胞)和骨细胞中。为了分离成骨细胞和成骨细胞，将DMP1-TPZ转基因小鼠(骨细胞特异性标记)与pOBCol2.3GFP-sapirre小鼠(转基因活跃于成骨细胞和骨细胞)杂交。酶促释放的头盖骨细胞将在双色系统的基础上进行荧光分选，以获得分离的成骨细胞和骨细胞群。除了基因表达分析，这项研究还将探讨在骨细胞阶段高表达并有可能在骨细胞生物学中发挥重要作用的基因的作用。对它们基因表达谱的分析将为更好地理解调节骨量的机制提供重要信息。我们将通过siRNA评估逆转录病毒过表达或下调目的基因的效果。这项研究的结果将为一项更大的提案奠定基础，该提案将包括测试该分析确定的特定基因的缺乏或过度表达在体内的影响。

DESCRIPTION (provided by applicant): Osteocytes are terminally differentiated cells derived from osteoblasts that become entrapped in their own matrix. Osteocytes survive by establishing a mutually connected canalicular network of dendritic processes by which they communicate with other osteocytes and other osteoblast lineage cells. It is well known that bone tissue has the capacity to alter its mass and structure in response to mechanical strain using osteocytes as mechanosensory cells. Identification of genes involved in these processes requires isolation of a pure population of osteocytes and a comparison to the gene expression profile to cells at other maturation stages of the osteoblast lineage. In this project we propose to evaluate the gene expression profile of an isolated osteocyte population and contrast its gene expression to osteoblasts as they transition into the osteocyte stage. A defined model of mouse calvarial osteoblast differentiation during the process of intramembranous ossification will be utilized. Cells of all maturation stages will be isolated using a sequential enzymatic digestion of parietal bones derived from mice harboring GFPtopaz driven by the dentin matrix protein 1 (DMP-1) promoter. This promoter has been shown to drive the expression of GFP to cells that are becoming entrapped in the matrix (preosteocytes) and in the osteocytes. To separate osteocytes from osteoblasts, DMP1-tpz transgenic mice (osteocyte specific marker) will be crossed with pOBCol2.3GFP-saphirre mice (transgene active in osteoblasts and osteocytes). Enzymatically released cells from calvaria will be fluorescence-sorted to obtain isolated populations of osteoblasts and osteocytes on the basis of the two-color system. In addition to gene expression analysis, this study will address the role of genes that are highly expressed at the osteocyte stage and have the potential to play an important role in osteocyte biology. The analysis of their gene expression profile will provide important information for a better understanding of the mechanisms that regulate bone mass. We will evaluate the effects of retroviral overexpression or downregulation of genes of interest by siRNA. The results of this study will generate the basis for a larger proposal that will include testing the in vivo effects of deficiency or overexpression of particular genes identified by this analysis.