CHARACTERIZATION OF A HUMAN OSTEOCLAST-LIKE CELL LINE

人类破骨细胞样细胞系的表征

• 批准号: 3157307
• 负责人: JAMES F DUNN
• 金额: $ 9.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-20 至 1988-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3157307
• 关键词: 1,25 dihydroxycholecalciferol; affinity chromatography; azacitidine; biological models; calcitonin; cell bank /registry; cell differentiation; colony stimulating factor; cytogenetics; gene expression; genetic strain; hormone regulation /control mechanism; monoclonal antibody; osteoblasts; osteoclast activating factor; osteoclasts; osteosarcoma; parathyroid hormones; physiologic bone resorption; retinoate

Much information has been learned about the osteoblast through the use of several cloned osteosarcoma cell lines which have the osteoblast phenotype and have provided convenient models of osteoblast behavior and function. No such cell line has been available as a model for the osteoclast. In part, this may be because the mature osteoclast is a terminal cell which does not proliferate. I have cloned a novel non-tumor forming cell line (FM-2) with osteolytic properties from a human osteosarcoma. Phenotypic characterization of this cell line as expressed in their ultrastructure, enzyme contents, multinucleation, calcitonin receptors (see Appendix) suggests that it is either an osteoclast precursor or a unique bone resorbing cell. The formation of spleen colony forming units when infused into irradiated rats and the phenotypic modulations shown by this cell when grown on three-dimensional collagen lattices further suggest that it may be an osteoclast "stem cell". The overall objective of this research proposal is to determine if it is a cell in the osteoclast lineage, to further characterize the osteoclastic properties of the FM-2 cell line and to define the conditions which permit maximal expression of these osteoclast-like properties. The experimental protocol is designed to: 1) identify and optimize the culture and substrate conditions which are critical for differentiation of the FM-2 cell line. This will be achieved by culturing the cells on devitalized bone or reconstituted substrates which may resemble bone or in an isolated in vivo system, the atriocular space of nude mice. A morphologic and biochemical profile to monitor phenotypic changes in the line or new sublines will be developed, 2) determine the effects of hormones active in bone (e.g., parathyroid hormone, calcitonin, or 1,25(OH)2D3) and differentiation factors (e.g., colony stimulating factor, OAF, 5-azacytidine, retinoic acid) on the phenotypic expression of the cell line, 3) restore bone remodeling function to microphthalmic mice by FM-2 cell grafts, 4) isolate and partially purify the bone resorbing factor using hydroxyapatite chromatography. The partially purified factor will be used to generate monoclonal antibodies for subsequent use in affinity chromatography and studies of the biological properties of the factor. These studies should provide new information on the differentiation of bone resorbing stem cells in vitro. The establishment of differentiated bone resorbing cells may prove useful as modes for understanding the biology and biochemistry of osteoclasts and other bone resorbing cells. Such models could also be applied to investigations of the mechanisms of bone loss in both physiologic and pathologic states.

许多关于成骨细胞的信息都是通过使用 几个具有成骨细胞表型的克隆骨肉瘤细胞系 为研究成骨细胞的行为和功能提供了方便的模型。 目前还没有这样的细胞系作为破骨细胞的模型。在……里面 这可能是因为成熟的破骨细胞是一个终末细胞，它 不会扩散。我克隆了一种新的非肿瘤形成细胞系 (FM-2)，具有来自人类骨肉瘤的溶骨特性。表型 在其超微结构中表达的这种细胞系的特征， 酶含量、多核、降钙素受体(见附录) 表明它要么是破骨细胞前体，要么是一种独特的骨骼 吸血细胞。输液时脾集落形成单位的形成 转化为受辐射的大鼠和这种细胞表现出的表型调节 生长在三维胶原晶格上的进一步表明，它可能是 一种破骨细胞“干细胞”。这项研究提案的总体目标是 是确定它是否是破骨细胞谱系中的细胞，以进一步 鉴定FM-2细胞系的破骨细胞特性 定义允许最大限度地表达这些内容的条件 破骨细胞样特性。 实验协议的设计目的是：1)识别和优化 对分化至关重要的培养和底物条件 FM-2细胞系。这将通过将细胞培养在 失活的骨或重建的底物，可能类似骨或在 一个孤立的活体系统，裸鼠的房眼空间。一个 用于监测表型变化的形态和生化特征 将开发线路或新的子线路，2)确定 骨骼中活跃的激素(如甲状旁腺激素、降钙素或 1，25(OH)2D3)和分化因子(例如，集落刺激因子， Oaf、5-氮胞苷、维甲酸)对细胞表型表达的影响 LINE，3)FM-2恢复小鼠眼部骨重建功能 细胞移植，4)分离和部分纯化骨吸收因子 使用羟基磷灰石层析。部分纯化的因子将是 用于产生单抗以供后续亲和使用 对该因子的生物学性质进行了层析和研究。 这些研究将为骨的分化提供新的信息。 在体外吸收干细胞。分化骨的建立 重新吸收细胞可能被证明是有用的，作为理解生物学和 破骨细胞和其他骨吸收细胞的生物化学。这样的模型 也可应用于骨丢失机制的研究 无论是生理状态还是病理状态。