Molecular analysis of abnormal fracture healing process

采用原位杂交等分子组织学技术研究骨折愈合受损过程。用ODS（Osteogenesis Disorder Shionogi）大鼠建立坏血病模型。维生素C的耗竭强烈抑制了骨折愈合中的膜骨化过程。特别是，检测不到骨桥蛋白（位于骨基质中的细胞外基质蛋白）的表达。软骨内骨化的早期阶段没有受到强烈的抑制。维生素C缺乏大鼠的软骨细胞数量与正常大鼠相当。然而，只有少数肥大的软骨细胞表达骨桥蛋白nRNA的观察。结果表明，由于骨桥蛋白的表达受损，在坏血病的钙化过程被强烈抑制的可能性。此外，在体内和体外的骨桥蛋白基因的表达调控转录因子进行了鉴定。CBFA 1、PU. 1、MITF与骨桥蛋白基因5 '侧翼区协同作用。cbfa-1基因敲除小鼠未检测到骨桥蛋白基因的表达。这种基因敲除小鼠的骨组织几乎没有钙化。骨桥蛋白参与了钙化过程。骨桥蛋白在骨重建中的生物学作用也进行了研究。骨细胞受到“压”的机械应力后，骨视素基因的表达增强。结果表明，骨细胞通过表达骨桥蛋白基因而对机械应力产生敏感反应。

Impaired fracture healing process was investigated by molecular histological techniques such as in situ hybridization. ODS (Osteogenesis Disorder Shionogi) rat was used for Scurvy model. Membranous ossification process in fracture healing was strongly inhibited by the depletion of vitamin C.Particularly, no expression of osteopontin, extracellular matrix protein located in the bone matrix, was detected. Early stages of endochondral ossification was not strongly inhibited. The unmber of chondrocytes in vitamin C depleted rats was comparable to that of normal rat. However, only a few number of hypertrophic chondrocytes which express osteopontin nRNA was observed. The results suggest the possibility that due to the impaired expression of osteopontin, calcification process in the scurvy was strongly inhibited. Furthermore, transcriptional factors regulating the expression of osteopontin gene was identified in vivo and in vitro. CBFA1, PU.1, MITF were cooperatively interacted with 5'-flanking region of osteopontin gene. No expression of osteopontin gene was detected in the knockout mice of cbfa-1 gene. This knockout mice showed almost no calcification in the bone tissue. The result also suggested the involvement of osteopontin to the calcification process. The biological role of osteopontin for the bone remodeling was also investigated. Enhanced expression of osteopnotin gene was observed by mechanical stress of "press" in the osteocytes. The result indicated that the osteocytes acted as sensory cells respond to echanical stress by espressing osteopontin gene.