IDENTIFYING COLLAGEN-I-INDUCED MRNAS IN OSTEOBLASTS

慢性骨量疾病是一个日益严重的医疗保健问题 这就需要改进诊断和治疗。重要的是 细胞外基质(ECM)成分，如I型胶原到 骨骼的生理学是通过在 成骨不全和绝经后骨质疏松症的个体。 然而，ECM介导的骨生理调节还没有很好的定义。 细胞外基质-成骨细胞的相互作用可能是骨肉瘤病理生理学的中心环节。 骨量疾病。本提案的目标是确定 成骨细胞1型胶原的末端反应(诱导蛋白表达) 互动。 这一目标将通过克隆成骨细胞培养来实现。 模型筛选及消减后的cDNA文库鉴定 CDNA(代表I型胶原诱导的蛋白)在 I型胶原刺激的OB。因此，这一行动的第一个具体目标是 提案将是创建代表光谱的cDNA库 由a)静息OB和b)I型胶原刺激的OB表达的蛋白质。这个 第二个特定目标是识别编码其蛋白的cDNA 表达是I型胶原刺激的OB特异的，需要两个cDNA 用于a)通过苯酚再结合合成消减探针的文库 杂交技术(PERT)和b)筛选I型胶原刺激 OB基因文库。最后，第三个目标将是确保 确定的蛋白质是OB与I型胶原相互作用的结果 矩阵。这将通过Northern印迹比较来实现 转录本的非刺激和I型胶原刺激的OB表达 确定为特定目的中的cDNA 2。 最近的一项关于骨重建的综述表明，骨重建的改善 骨量的临床处理将需要识别 骨重塑的分子组成。拟议的身份识别 ECM刺激的OB表达的蛋白质代表着一种 寻求确定成骨细胞分子决定因素的研究计划 生理和骨量。

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.