CHARACTERIZATION OF A HUMAN OSTEOCLAST-LIKE CELL LINE

人类破骨细胞样细胞系的表征

• 批准号: 3157306
• 负责人: JAMES F DUNN
• 金额: $ 8.11万
• 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/3157306
• 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是通过使用 几个克隆的具有成骨细胞表型的骨肉瘤细胞系 并提供了成骨细胞行为和功能的方便模型。 没有这样的细胞系可用作破骨细胞的模型。 在 这可能是因为成熟的破骨细胞是一种终末细胞， 不会扩散。 我克隆了一种新的非肿瘤形成细胞系 （FM-2）具有溶骨特性。 表型 该细胞系的超微结构表征， 酶含量、多核化、降钙素受体（见附录） 表明它要么是破骨细胞的前体， 再吸收细胞 输注时脾集落形成单位的形成 辐射大鼠和表型调制显示，这种细胞时， 在三维胶原晶格上生长的细胞进一步表明， 破骨细胞"干细胞"。 本研究提案的总体目标 是确定它是否是破骨细胞谱系中的细胞， 表征FM-2细胞系的骨细胞特性， 定义允许这些最大表达的条件 破骨细胞样特性。 实验方案被设计为：1）识别和优化 培养和基质条件是分化的关键 FM-2细胞系。 这将通过将细胞培养在 失活骨或类似骨的重组基质，或 一个孤立的体内系统，裸鼠的心房空间。 一 形态学和生物化学特征，以监测 线或新的子线将被开发，2）确定的影响， 在骨中有活性的激素（例如，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-氮杂胞苷、视黄酸）对细胞表型表达的影响 线，3）通过FM-2恢复小眼小鼠的骨重建功能 细胞移植物，4）分离并部分纯化骨吸收因子 使用羟基磷灰石色谱法。 部分纯化的因子将是 用于产生单克隆抗体，随后用于亲和层析 层析和生物学特性的研究。 这些研究将为骨的分化提供新的信息 体外吸收干细胞。 分化骨的建立 再吸收细胞可能被证明是有用的模式，为了解生物学和 破骨细胞和其他骨吸收细胞的生物化学。 这样的模型 也可用于研究骨丢失的机制， 生理和病理状态。