SEX STEROIDS, GROWTH FACTORS AND BONE CELL FUNCTION

这一应用的主要焦点将是使用培养的正常人 成骨样细胞(HOB)作为模型系统来理解 对正常成骨细胞功能的调节，并提供对 骨质疏松症的细胞功能异常。研究开发一种明确的、 自那以后，将继续进行无血清培养，以实现最佳细胞生长 培养基应1)促进这些缓慢细胞的产生 增长、劳动密集型和成本高昂，以及2)允许更准确 评估个别生长相关药物对这些指标的影响 细胞。我们目前的数据显示雌激素受体的存在。 在HOB细胞中存在雄激素和 黄体酮受体。我们计划继续分析 功能性类固醇受体在HOB细胞中的表达 骨肉瘤细胞系使用核结合试验、木炭试验、 Northern印迹(信使核糖核酸)和蛋白质印迹(蛋白质)分析。我们有 获得多种用于骨蛋白、生长的mRNAs的cDNA探针 因子、原癌基因和其他类固醇调节基因，包括 类固醇受体。我们计划筛选这些cdna探针，以选择那些 表现出最佳的雌激素调节。然后，使用这些cDNA，我们 将更详细地评估雌激素、雄激素和 孕激素对HOB细胞和部分骨肉瘤组织中mRNA水平的影响 细胞系。类固醇激动剂及其与异性的拮抗作用研究 类固醇和其他激素(甲状旁腺素、1，25二羟基维生素D3和 糖皮质激素)将在HOB细胞中进行。类固醇对人体的作用 骨蛋白质的水平将在类固醇的例子中被测量。 这些重要基因的表达调控发生在 翻译/翻译后的级别。最后，我们计划继续我们的 性激素对小鼠生殖功能影响的研究 转化生长因子(转化生长因子α、转化生长因子β)与胰岛素样生长 HOB细胞中的因子(IGF-I和IGF-II)在mRNA，蛋白质， 和活动。与格雷格·芒迪博士和同事的合作， 德克萨斯大学圣安东尼奥分校将研究 雌激素与性激素和甲状旁腺素的相互作用 影响转化生长因子-β的产生。同样在相同的合作中，效果 雌激素处理的HOB细胞条件培养液对破骨细胞的影响 将对功能(骨吸收)进行调查。希望这一天 上述研究将对骨骼调节途径有深入的了解 并最终导致这些[进化通路]的异常 骨质疏松症导致了更好的治疗策略。

The major focus of this application will be to use cultured normal human osteoblast-like (hOB) cells as a model system for understanding the regulation of normal osteoblast function and to provide insights on abnormal cell function in osteoporosis. Studies to develop a defined, serum-free medium for optimal cell growth will be continued since this medium should 1) enhance the production of these cells which are slow growing, labor intensive and costly, and 2) permit more accurate assessment of the effects of individual growth related agents on these cells. Our present data demonstrate the presence of estrogen receptors in hOB cells with indications of the presence of androgen and progesterone receptors. We plan to continue our analyses of the functional sex steroid receptors in the hOB cells and selected osteosarcoma lines using a nuclear binding assay, a charcoal assay, Northern blot (mRNA), and Western blot (protein) analyses. We have obtained cDNA probes to a variety of mRNAs for bone proteins, growth factors, proto-oncogenes and other steroid regulated genes including steroid receptors. We plan to screen these cDNA probes to select those which display optimal estrogen regulation. Then, using these cDNAs, we will assess in greater detail the effects of estrogens, androgens and progestins on the mRNA levels in hOB cells and in selected osteosarcoma cell lines. Studies of steroid agonism and antagonism with other sex steroids and other hormones (PTH, 1,25 dihydroxyvitamin D3, and glucocorticoids) will be performed in hOB cells. The steroid action on the levels of bone proteins will be measured in the instance the steroid regulation of the expression of these important genes occurs at the level of translation/post-translation. Finally, we plan to continue our investigations on the effects of sex steroids on the production of the transforming growth factors (TGF-a, TGF-b) and insulin-like growth factors (IGF-I adn IGF-II) in hOB cells at the level of mRNA, protein, and activity. Collaborations with Dr. Greg Mundy and coworkers, University of Texans at San Antonio, will involve studies of the interactions of estrogens with our sex steroids and with PTH to influence TGF-B production. Also in the same collaboration, the effects of conditioned media from estrogen treated hOB cells on osteoclast cell function (bone resorption) will be investigated. It is hoped that the above investigations will yield insights into bone regulatory pathways and ultimately into abnormalities of these [pathways in involutional osteoporosis resulting in better strategies for its treatment.