Resolution of Stage-Specific Growth Plate Chondrocytes

阶段特异性生长板软骨细胞的分辨率

• 批准号: 6804404
• 负责人: Wenhan Chang
• 金额: $ 8.25万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-25 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6804404
• 关键词: biological signal transduction; bone development; calcium flux; cell differentiation; cell growth regulation; cell sorting; chondrocytes; collagen; cyclic AMP; enzyme activity; flow cytometry; gene expression; genetic markers; genetic promoter element; laboratory mouse; osteopontin; parathyroid hormone related protein; phospholipase C; polymerase chain reaction; tissue /cell culture

DESCRIPTION (provided by applicant): Chondrocytes in the growth plate progress through steps of resting, proliferation, maturation, and hypertrophy and finally become terminally differentiated cells that produce a mature mineralized matrix and support bone formation. As differentiation proceeds, chondrocytes express unique marker genes indicative of specific stages of differentiation. The cellular and molecular bases underlying the steps in growth plate differentiation remain largely unknown. This is largely due to a lack of information on the functional properties of chondrocytes at each different stage. A major obstacle in obtaining this information is the heterogeneous nature of growth plate chondrocytes. Density gradient centrifugation has been used to separate these chondrocytes into subpopulations, but it is unclear that differences in size and density accurately reflect stages of chondrocyte differentiation. This technique also requires starting with a large number of cells. This is not easily adapted to the mouse growth plate, and there are numerous transgenic and gene "knock-out" mice with striking growth plate abnormalities. It would be ideal to have a technique that could reliably separate out functionally distinct populations of chondrocytes from the mouse growth plate. We propose to develop a cell-sorting technique based on the hypothesis that differentiation stagespecific growth plate chondrocytes can be identified and isolated according to the promoter activities of the marker genes expressed at discrete stages of differentiation. We will also test the hypothesis that the signal transduction pathways that are active in chondrocytes change with the stage of differentiation. In Aim 1 we will express the promoter elements for the type II collagen (alpha1subunit, cyclin D1, type X collagen alpha1 subunit, and osteopontin genes, which are pre-tagged with distinct fluorescent protein cDNAs in chondrocytes using replication-deficient adenoviruses. We will sort by FACS different subpopulations of chondrocytes according to the level of activation of promoters, reflected in different fluorescent signal intensities. In Aim 2, we will determine whether the profile of signal transduction responses to parathyroid hormone related protein (PTHrP), an important modulator of chondrocyte differentiation, differs in chondrocytes at different stages of differentiation. The effects of PTHrP on cyclic AMP (cAMP) formation, phospholipase C (PLC) activity, and intracellular Ca 2. mobilization will be examined in different subpopulations of chondrocytes.

描述（申请人提供）：生长板中的软骨细胞经历静息、增殖、成熟和肥大等步骤，最终成为终末分化细胞，产生成熟的矿化基质并支持骨形成。随着分化的进行，软骨细胞表达指示特定分化阶段的独特标记基因。生长板分化步骤背后的细胞和分子基础仍然很大程度上未知。这很大程度上是由于缺乏有关软骨细胞在每个不同阶段的功能特性的信息。获得此信息的一个主要障碍是生长板软骨细胞的异质性。密度梯度离心已用于将这些软骨细胞分离成亚群，但尚不清楚大小和密度的差异能否准确反映软骨细胞分化的阶段。该技术还需要从大量细胞开始。这不容易适应小鼠生长板，并且有许多转基因和基因“敲除”小鼠具有明显的生长板异常。拥有一种能够可靠地从小鼠生长板中分离出功能不同的软骨细胞群的技术将是理想的。我们建议开发一种细胞分选技术，该技术基于以下假设：可以根据在不同分化阶段表达的标记基因的启动子活性来鉴定和分离分化阶段特异性生长板软骨细胞。我们还将检验软骨细胞中活跃的信号转导途径随着分化阶段而变化的假设。在目标 1 中，我们将表达 II 型胶原蛋白的启动子元件（α1 亚基、细胞周期蛋白 D1、X 型胶原蛋白 α1 亚基和骨桥蛋白基因，这些元件使用复制缺陷型腺病毒在软骨细胞中预先标记有不同的荧光蛋白 cDNA。我们将根据目标 2 中反映的不同荧光信号强度，通过 FACS 对不同的软骨细胞亚群进行排序。确定软骨细胞分化的重要调节剂甲状旁腺激素相关蛋白 (PTHrP) 的信号转导反应在不同分化阶段的软骨细胞中是否存在差异 PTHrP 对环 AMP (cAMP) 形成、磷脂酶 C (PLC) 的影响。将在不同的软骨细胞亚群中检查Ca 2. 活性和细胞内Ca 2. 动员。