ESTROGEN RECEPTOR STRUCTURE/FUNCTION IN AGING BONE

拟议研究的长期目标是确定 重组人胰岛素的生物学功能及其分子机制 如生长因子-I(rhIGF-I)。胰岛素样生长因子-I(IGF-I)是 一种具有调节成骨细胞潜能的多肽 功能。建议的研究是临床研究的结果。 旨在确定重组人胰岛素样生长因子-I和雌激素对骨骼的影响 绝经后妇女的更年期。为了了解它们的机制 参与IGF-I对骨骼的作用，首席调查员建议 重组人胰岛素样生长因子-I对细胞外基质代谢的影响 体外培养的人成骨细胞。具体地说，要衡量 胰岛素样生长因子-I对细胞外基质基因表达和蛋白水平的影响 组分，骨细胞IGF-I反应作为年龄的函数 基质的耐心和矿化状态、稳定性和能力 作为矿化的脚手架的基质，以及 生长激素(GH)、雌激素和甲状旁腺激素(PTH)对骨骼的影响 细胞对IGF-I的反应。 I型骨基质蛋白和mRNA的稳态水平 胶原蛋白、蛋白多聚糖和粘附素、骨连接蛋白和 将分析碱性磷酸酶以确定IGF-I的作用 关于骨细胞基质的组成。蛋白质和信使核糖核酸水平 由首席调查员开发的技术确定，或 在人成骨细胞外基质研究中的应用 新陈代谢。骨细胞时间序列的影响 培养中的发展，无论是前矿物质还是矿化 细胞外基质对IGF-I的反应性将通过 青壮年(10-18岁)稳态基质成分比较 成人(50岁以上)人骨小梁细胞培养。这个 细胞外基质的稳定性将通过放射性标记进行评估 并对这些特定的骨基质成分进行脉冲追逐分析。这个 胰岛素样生长因子-I对基质矿化能力的影响 沉积将通过确定体外时间框架进行评估 通过分析矿物质含量和蛋白质含量 (包括骨钙素)的细胞外基质。内分泌 人骨细胞对IGF-I反应性的调节 通过表征生长激素、雌激素和甲状旁腺素对 基质含量(稳态放射性标记)、稳定性(脉冲追逐 放射性标记)和成矿能力。的方法 检测细胞外基质成分和稳定性及 IGF-I治疗和全身因子对矿化的响应 调控将定义体外骨蛋白的整合模式 蛋白多糖代谢和骨矿化。

The long term objective of the proposed research is to determine the biological function and molecular mechanism of recombinant human insulin like growth factor-I (rhIGF-I). Insulin like growth factor-I (IGF-I) is a polypeptide that has the potential to modulate osteoblast cell function. The proposed studies are an outgrowth of clinical studies designed to determine the effects of rhIGF-I and estrogen on skeletal turnover in postmenopausal women. In order to understand the mechanisms involved in IGF-I action on bone, the Principal Investigator proposes to study the effects of rhIGF-I on the extracellular matrix metabolism of human osteoblasts in vitro. Specifically, to measure the effect of IGF-I on mRNA expression and protein levels of extracellular matrix components, bone cell IGF-I response as a function of the age of the patient and mineralized state of the matrix, stability and competency of the matrix as a scaffolding for mineralization, and the influence of growth hormone (GH), estrogen, and parathyroid hormone (PTH) on bone cell response to IGF-I. The steady-state levels of bone matrix protein and mRNA for type I collagen, the proteoglycans biglycan and decorin, osteonectin and alkaline phosphatase will be analyzed to determine the effect of IGF-I on the composition of the bone cell matrix. Protein and mRNA levels will be determined by techniques developed by the Principal Investigator or utilized in prior studies of human osteoblast extracellular matrix metabolism. The effect of the temporal sequence of bone cell development in culture, either a pre-mineral or a mineralizing extracellular matrix, on IGF-I responsiveness will be assessed by comparing steady-state matrix composition for young adult (10-18 years) and adult (over 50 years) human trabecular bone cell cultures. The stability of the extracellular matrix will be assessed by radiolabeling and pulse-chase analysis of these specific bone matrix components. The effect of IGF-I on the competency of the matrix as a site for mineral deposition will be assessed by determining both the in vitro time frame of mineralization and by analyzing the mineral content and protein (including osteocalcin) of the extracellular matrix. The endocrine modulation of human bone cell responsiveness to IGF-I will be investigated by characterizing the effect of GH, estrogen, and PTH on matrix content (steady-state radiolabeling), stability (pulse-chase radiolabeling), and competency for mineralization. The approach of assaying extracellular matrix composition and stability and mineralization in response to IGF-I treatment and systemic factor modulation will define an integrated pattern of in vitro bone protein and proteoglycan metabolism and bone mineralization.