IDENTIFICATION OF DIFFERENTIALLY EXPRESSED CRITICAL GENES IN CHONDROCLASTS AND OS

描述（由申请人提供）：在软骨内成骨过程中，矿化软骨基质在骨形成和重塑之前被吸收。虽然这种软骨基质吸收是由表现出许多破骨细胞表型特征的细胞介导的，但一个长期存在的问题是，这些破软骨细胞和更好表征的骨吸收破骨细胞之间的基因表达是否存在差异。为了解决这个问题，我们将使用我们的小鼠股骨骨折模型，在愈合的后期阶段，早期骨痂中的钙化软骨基质将在骨痂重塑之前被重塑。虽然组织学证据显示了破软骨细胞和破骨细胞的不同特征，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阳性细胞再吸收矿化软骨基质（破软骨细胞）和细胞再吸收骨基质（破骨细胞）的基因表达谱。我们将进一步确定哪些差异表达的基因对软骨细胞和破骨细胞的各自功能是重要的。这些研究将在从骨折小鼠骨骼的组织学样本中取出的细胞上进行。我们认为，软骨细胞和破骨细胞周围的基质和环境严重影响这些细胞的表达谱，这些细胞几乎肯定来自一个共同的祖细胞。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基因表达谱的差异。我们的中心假设假设破骨细胞和破软骨细胞具有不同的基因表达模式，这促进了它们独特的功能。我们提出以下两个特定目的来验证这一假设：特定目的1：研究在小鼠骨折愈合过程中，激光捕获的trap阳性细胞重新吸收软骨或骨基质之间的分子差异。我们假设重新吸收钙化软骨基质的trap阳性细胞与重新吸收骨基质的trap阳性细胞表现出不同的基因表达谱。特定目的2：建立从骨折股骨中分离的破骨细胞和软骨细胞的功能差异。我们假设破骨细胞和破软骨细胞之间存在促进基质特异性吸收的功能差异。我们提出的研究将首次利用骨折愈合小鼠模型来证明，在软骨内成骨过程中，调节软骨和骨重塑的分子机制是否存在功能差异。

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.