IDENTIFICATION OF DIFFERENTIALLY EXPRESSED CRITICAL GENES IN CHONDROCLASTS AND OS

描述(申请人提供)：在软骨内骨化过程中，矿化的软骨基质在骨形成和重塑之前被吸收。虽然这种软骨基质的吸收是由表现出许多破骨细胞表型特征的细胞介导的，但这些软骨破骨细胞与特征更好的骨吸收破骨细胞之间的基因表达是否存在差异，仍然是一个长期存在的问题。为了解决这个问题，我们将使用我们的小鼠股骨骨折模型，在愈合的后期，骨痂早期的钙化软骨基质将在骨痂重塑之前重塑。虽然组织学证据显示软骨破骨细胞和破骨细胞有明显的相同之处，但关于区分它们各自功能的分子特征的信息很少。我们的目标是比较TRAP阳性细胞吸收矿化软骨基质(软骨破骨细胞)和吸收骨基质细胞(破骨细胞)的基因表达谱。我们将进一步确定这些差异表达基因中哪些对于软骨细胞和破骨细胞各自的功能是重要的。这些研究将在从骨折小鼠骨骼的组织学样本中提取的细胞上进行。我们认为，软骨破骨细胞和破骨细胞周围的基质和环境对这些细胞的表达谱有重要影响，几乎可以肯定这些细胞来自共同的祖细胞。因此，我们设计了我们的研究来检查直接从体内环境中移除的细胞中基因表达的差异，以便我们能够最好地了解它们的基因表达谱中的差异。我们的中心假设是破骨细胞和软骨破骨细胞具有不同的基因表达模式，这有助于它们具有独特的功能。我们提出了以下两个特定的目的来验证这一假设：特定的目的1：检查在小鼠骨折愈合过程中吸收软骨或骨基质的激光捕获TRAP阳性细胞之间的分子差异。我们假设，吸收钙化软骨基质的TRAP阳性细胞将表现出与吸收骨基质的TRAP阳性细胞不同的基因表达谱。目的：建立分离自股骨的破骨细胞和软骨破骨细胞之间的功能差异。我们假设破骨细胞和软骨破骨细胞之间存在功能差异，这促进了基质特异性的吸收。我们提出的研究将首次使用骨折愈合的小鼠模型，证明在出生后软骨内骨化过程中，调控软骨和骨重建的分子机制是否存在功能差异。 公共卫生相关性：长骨的骨骼发育和骨折修复需要连续的细胞事件，这些事件不仅涉及软骨和骨细胞，而且还涉及重塑软骨(软骨破骨细胞)和骨(破骨细胞)基质的细胞。在这里，我们建议确定这两种细胞类型之间的分子差异，并进一步研究软骨破损细胞介导软骨基质重塑以正确愈合骨折所需的关键基因。越来越多的临床证据表明，软骨基质重塑对于骨骼的正常发育和正常的骨折愈合非常重要。从这些研究中获得的数据可能有助于开发新的治疗方式，以加速年轻人和老年人的骨折修复。

DESCRIPTION (provided by applicant): During endochondral ossification, mineralized cartilage matrix is resorbed prior to bone formation and remodeling. While this cartilage matrix resorption is mediated by cells that exhibit many phenotypic features of osteoclasts, a longstanding question remains as to whether differences exist in the gene expression between these chondroclasts and the better characterized bone resorbing osteoclasts. To address this question we will use our mouse femoral fracture model, in which calcified cartilage matrices in the early callus will be remodeled prior to the remodeling of bony callus, during late stages of healing. While histological evidence has shown distinct identities of chondroclasts and osteoclasts, little information is available regarding the molecular characteristics that differentiate their respective functions. Our goal is to compare the gene expression profiles of TRAP-positive cells resorbing mineralized cartilage matrix (chondroclasts) with that of cells resorbing bone matrix (osteoclasts). We will further determine which of these differentially expressed genes is important for the respective functions of chondrocytes versus osteoclasts. These studies will be performed on cells that are removed from histologic samples of fractured mouse bones. We believe that the matrix and environment surrounding chondroclasts and osteoclasts critically influences the expression profiles of these cells, which almost certainly derive from a common progenitor. Therefore, we have designed our studies to examine differences in gene expression in cells that are removed directly from their in vivo environment so that we can best appreciate differences in their gene expression profiles. Our central hypothesis posits that osteoclasts and chondroclasts have distinct patterns of gene expression, which facilitate their unique functions. We propose the following two Specific Aims to test this hypothesis: Specific Aim1: To examine the molecular differences between laser-captured TRAP-positive cells resorbing cartilage or bone matrices during fracture healing in mice. We hypothesize that TRAP-positive cells resorbing calcified cartilage matrix will exhibit a different gene expression profile than TRAP-positive cells resorbing bone matrix. Specific Aim2: To establish functional differences between chondroclasts and osteoclasts isolated from fractured femurs. We hypothesize that functional differences exist between osteoclast and chondroclasts, which facilitate matrix-specific resorption. Our proposed studies will for the first time demonstrate, using a fracture healing mouse model, whether there are functional differences in the molecular mechanisms regulating cartilage versus bone remodeling during endochondral ossification post-natally. PUBLIC HEALTH RELEVANCE: Skeletal development and fracture repair of long bones require successive cellular events that not only involve cartilage and bone cells, but also cells that remodel cartilage (chondroclasts) and bone (osteoclasts) matrices. Here we propose to identify molecular differences between these two cell types and further examine the critical genes that are required for chondroclasts to mediate cartilage matrix remodeling for proper fracture healing. Clinical evidence is emerging demonstrating the importance of cartilage matrix remodeling for proper skeletal development and normal fracture healing. Data obtained from these studies may help develop novel therapeutic modalities to accelerate fracture repair in young and aged populations.