Molecular analysis of abnormal fracture healing process

采用原位杂交等分子组织学技术对骨折愈合过程进行研究。以成骨障碍Shionogi大鼠为坏疽模型。维生素C的缺乏强烈抑制了骨折愈合过程中的膜性成骨过程，尤其是未检测到位于骨基质中的骨桥蛋白的表达。早期软骨内骨化的抑制作用不强。维生素C耗竭大鼠的软骨细胞数与正常大鼠相当。只有少数肥大的软骨细胞表达骨桥蛋白nRNA。结果提示，由于骨桥蛋白的表达受损，坏疽的钙化过程可能受到强烈的抑制。此外，还在体内和体外鉴定了调控骨桥蛋白基因表达的转录因子。CBFA1、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。这种基因敲除的小鼠在骨组织中几乎没有钙化。这一结果还提示骨桥蛋白参与了钙化过程。并探讨了骨桥蛋白在骨重建中的生物学作用。骨桥蛋白基因的表达可通过“压”力在骨细胞中得到增强。结果表明，骨细胞作为感觉细胞通过表达骨桥蛋白基因对机械应力做出反应。

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.