IDENTIFYING COLLAGEN-I-INDUCED MRNAS IN OSTEOBLASTS

鉴定成骨细胞中 I 型胶原诱导的 MRNAS

• 批准号: 3425965
• 负责人: LYNDON F COOPER
• 金额: $ 4.14万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3425965
• 关键词: bone density; collagen; complementary DNA; extracellular matrix; extracellular matrix proteins; gel electrophoresis; gene expression; genetic library; messenger RNA; northern blottings; nucleic acid probes; nucleic acid sequence; osteoblasts; plasmids; southern blotting; subtraction hybridization; tissue /cell culture

Chronic diseases of bone mass represent a growing health care problem which demands improved diagnosis and treatment. The importance of extracellular matrix (ECM) components such as type I collagen to the physiology of bone is illustrated by the collagen mutations found in individuals with osteogenesis imperfecta and post menopausal osteoporosis. Yet, ECM-mediated regulation of osseous physiology is not well defined. ECM-ostoblast interactions may be central to the pathophysiology of diseases of bone mass. The goal of the present proposal is to identify end-responses (induced protein expression) of osteoblast -type 1 collagen interactions. This goal will be accomplished using a clonal osteoblast (OB) cell culture model and subtracted cDNA probe screening of a cDNA library to identify cDNAs (representing collagen I-induced proteins) uniquely present in collagen I-stimulated OB. Therefore, the first specific aim of this proposal will be to create cDNA libraries which represent the spectrum of proteins expressed by a) resting OB and b) collagen I-stimulated OB. The second specific aim, to identify the cDNAs that encode proteins whose expression is specific to collagen I-stimulated OB, will require both cDNA libraries to a) synthesize a subtracted probe by the Phenol Reassociation Hybridization Technique (pERT) and b)to screen the collagen I-stimulated OB cDNA library. Finally, the third aim will be to assure that the identified proteins are the result of OB interaction with a collagen I matrix. This will be accomplished by Northern blot comparison of unstimulated and collagen I-stimulated OB expression of transcripts identified as cDNAs in Specific Aim 2. A recent review of bone remodeling suggested that improvements in the clinical management of bone mass will require the identification of the molecular components of bone remodelling. The proposed identification of proteins expressed by ECM-stimulated OB represents the initiation of a research program that seeks to define molecular determinants of osteoblast physiology and bone mass.

骨量慢性疾病是一个日益严重的医疗保健问题 这需要改进诊断和治疗。的重要性 细胞外基质 (ECM) 成分，例如 I 型胶原蛋白 骨的生理学可以通过发现的胶原蛋白突变来说明 患有成骨不全和绝经后骨质疏松症的个体。 然而，ECM 介导的骨生理调节尚未明确。 ECM-成骨细胞相互作用可能是病理生理学的核心 骨量疾病。本提案的目标是确定 成骨细胞 1 型胶原的最终反应（诱导蛋白表达） 互动。 这一目标将通过克隆成骨细胞 (OB) 细胞培养来实现 模型和扣除 cDNA 探针筛选 cDNA 文库以鉴定 cDNA（代表 I 型胶原蛋白诱导蛋白）独特存在于 胶原蛋白 I 刺激的 OB。因此，本次活动的第一个具体目标 提议将创建代表谱系的 cDNA 文库 由 a) 静息 OB 和 b) 胶原蛋白 I 刺激的 OB 表达的蛋白质。这 第二个具体目标是鉴定编码蛋白质的 cDNA，其 表达对胶原蛋白 I 刺激的 OB 具有特异性，需要 cDNA a) 通过苯酚重联合成消减探针 杂交技术 (pERT) 和 b) 筛选 I 型胶原蛋白刺激 OB cDNA 文库。最后，第三个目标是确保 鉴定出的蛋白质是 OB 与 I 型胶原蛋白相互作用的结果 矩阵。 这将通过 Northern blot 比较来完成 未刺激和胶原蛋白 I 刺激的 OB 转录本表达 在具体目标 2 中被鉴定为 cDNA。 最近对骨重塑的综述表明，骨重建的改善 骨量的临床管理需要确定 骨重塑的分子成分。拟议的识别 ECM 刺激的 OB 表达的蛋白质代表了 旨在定义成骨细胞分子决定因素的研究计划 生理学和骨量。