OIM--A MURINE REPLICA OF HUMAN OSTEOGENESIS IMPERFECTA

成骨不全是一种遗传性结缔组织疾病 它主要影响骨骼。最近的调查表明， 大多数OI病例是影响新陈代谢的突变的结果 I型胶原蛋白的α1或α2链。这些突变 可能干扰了细胞外基质中胶原纤维的形成 和/或损害这些异常纤维与其他蛋白质的相互作用 和骨骼基质的蛋白多糖成分。OI也很重要 因为它可能提供对相关的病理生理学的洞察。 包括特发性骨质疏松症在内的骨骼形成障碍。这个 显示特定的I型胶原突变(点突变， 删除、插入)尚未阐明OI的病理生理学， 特别是关于两个重要问题；1)影响 正常骨骼形成的特定突变和2)因子 对在人类身上观察到的高度可变表现力负责 喂。一个可存活的OI动物复制品的可用性将极大地 促进对这些重要问题的调查。这个 成骨不全(OIM)小鼠是自发的、 在杰克逊一家的繁育群体中首次发现非致命性突变 实验室。这个突变体似乎具有生化和表型特征。 类似于一个经过充分研究的严重OI III型患者。像这个OI 患者，纯合子OIM小鼠的表型包括生长减少， 自发性骨折和渐进性骨骼变形，包括 脊柱侧弯和骨质疏松。纯合OIM真皮的生化研究 成纤维细胞，皮肤，骨骼，肌腱，心脏，肝脏和肾脏显示 未能合成α2(I)链。此外，我们最近定义了一个 改变Proalpha2(I)基因4060位的假定G缺失 COOH-前肽的最后49个氨基酸。因此，只有字母1(I)3 发现同源三聚体在骨架基质中积累。字母1(I)3 高三聚体胶原蛋白被结合到矿化的细胞外 然而，生成的骨架是疏骨性的，变形的并且更小 而不是野生型小鼠。这些数据，结合放射照片和 组织化学数据表明，高三聚体胶原不能提供 可在其上形成正常骨骼的合适模板。 我们建议1)通过测序相同的区域来验证可能的突变 在第二个纯合子OIM小鼠和杂合子OIM小鼠中，2) 维持一个能够产生足够动物的小鼠群体，3) 确定突变对骨骼形成的影响，4)培养颅骨 成骨细胞检测其形成矿化的能力，三维 细胞外基质，5)利用不同的 启动子：增强元件和全长小鼠α2(I)cDNA，6) 建立转基因成骨细胞培养和表达这些基因的小鼠 载体，以及7)评估转基因的骨骼、牙齿和其他组织 小鼠研究这些结构对α1(I)3合成的影响 同源三聚体与正常I型异源三聚体在体外对骨形成的影响 在活体内。最后，我们将尝试拯救纯合子OIM小鼠 提供人类OI的生化和表型复制品，将负担得起 研究I型胶原病理生理学的难得机会 新陈代谢及其与骨骼形成的关系。

Osteogenesis imperfecta (OI) is a heritable disorder of connective tissue which predominately affects bone. Recent investigations indicate that the majority of OI cases are the result of mutations affecting the metabolism of the alpha 1 or alpha 2 chains of type I collagen. These mutations presumably disrupt collagen fiber formation in the extracellular matrix and/or impair the interaction of these abnormal fibers with other protein and proteoglycan components of the skeletal matrix. OI is also important because it may provide insight into the pathophysiology of related disorders of skeletal formation including idiopathic osteoporosis. The demonstration of specific type I collagen mutations (point mutations, deletions, insertions) has not shed light on the pathophysiology of OI, specifically with regard to two important questions; 1) the impact of specific mutations on normal skeletal formation and 2) factors which are responsible for the high degree of variable expressivity observed in human OI. The availability of a viable animal replica of OI will enormously facilitate the investigation of these important questions. The Osteogenesis Imperfecta (oim) mouse is the result of a spontaneous, nonlethal mutation first observed in the breeding stock of The Jackson Laboratory. This mutant appears to be biochemically and phenotypically similar to a well studied patient with severe OI type III. Like this OI patient, the phenotype of homozygous oim mice includes diminished growth, spontaneous fractures and progressive skeletal deformation, including scoliosis and osteoporosis. Biochemical study of homozygous oim dermal fibroblasts, skin, bone, tendon, heart, liver and kidney demonstrated a failure to synthesize alpha 2(I) chains. Also, we recently defined a putative G deletion at position 4060 of the proalpha 2 (I) gene that alters the last 49 amino acids of the COOH-propeptide. Therefore, only alpha1(I)3 homotrimer was found to accumulate in the skeletal matrix. alpha1(I)3 homotrimeric collagen is incorporated into a mineralized extracellular matrix, however, the resultant skeleton is osteopenic, deformed and smaller than in wild type mice. These data, combined with the radiographic and histochemical data, suggest that homotrimeric collagen does not provide a suitable template on which to form normal bone. We propose to 1) verify the putative mutation by sequencing the same region in a second homozygous oim mouse and in a heterozygous oim mouse, 2) maintain a mouse colony capable of generating sufficient animals, 3) determine the mutation's effect on skeletal formation, 4) culture calvarial osteoblasts to examine their ability to form a mineralizing, 3-dimensional extracellular matrix, 5) develop alpha2(I) minigenes utilizing different promoter: enhance elements and a full-length murine alpha2(I) CDNA, 6) develop transgenic osteoblast cultures and mice with these expression vectors, and 7) evaluate skeletal, dental and other tissues in transgenic mice for the effect of these constructs on the synthesis of alpha 1(I)3 homotrimer versus normal type I heterotrimer on bone formation in vitro and in vivo. Finally, we will attempt to rescue homozygous oim mice should provide a biochemical and phenotypic replica of human OI that will afford a unique opportunity to study the pathophysiology of type I collagen metabolism, and its relationship to skeletal formation.