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分子机制 如生长因子-I（rhIGF-I）。胰岛素样生长因子-I（IGF-I）是 具有调节成骨细胞的潜力的多肽 功能 拟议的研究是临床研究的结果 目的是确定rhIGF-I和雌激素对骨骼肌的影响， 绝经后妇女的周转率。 为了了解这些机制 参与IGF-I对骨骼的作用，主要研究者建议 研究rhIGF-I对细胞外基质代谢的影响 体外培养的人成骨细胞。 具体来说，为了衡量 IGF-I对细胞外基质mRNA表达和蛋白水平的影响 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 基质作为矿化的支架，以及 生长激素（GH）、雌激素和甲状旁腺激素（PTH）对骨 细胞对IGF-I的反应。 骨基质蛋白和I型胶原mRNA的稳态水平 胶原蛋白、蛋白聚糖双糖链蛋白聚糖和核心蛋白聚糖、骨粘连蛋白和 将分析碱性磷酸酶以确定IGF-I的作用 对骨细胞基质的组成有影响。蛋白质和mRNA水平将 通过主要研究者开发的技术确定，或 用于人成骨细胞细胞外基质的先前研究 新陈代谢. 骨细胞的时序性影响 文化的发展，无论是前矿物或矿化 细胞外基质对IGF-I反应性的影响将通过 比较年轻成人（10-18岁）的稳态基质组成 和成人（50岁以上）人小梁骨细胞培养物。 的 细胞外基质的稳定性将通过放射性标记 和这些特定骨基质成分的脉冲追踪分析。 的 IGF-I对基质作为矿物质位点的能力的影响 沉积将通过确定体外时间框架和体外时间框架来评估。 并通过分析矿物质含量和蛋白质 （包括骨钙素）的细胞外基质。 内分泌 调节人骨细胞对IGF-I的反应性， 通过表征GH、雌激素和PTH对 基质含量（稳态放射性标记）、稳定性（脉冲追踪 放射性标记）和矿化能力。 的临近 测定细胞外基质组成和稳定性， 矿化对IGF-I治疗和全身因素的反应 调节将确定体外骨蛋白的整合模式 以及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.