大鼠胫骨近端骨骺损伤后骨桥形成分子机制的研究

Ossification Mechanism for Bone Bridge Formation After Physical Injury in Young Rats

利用胫骨近端干骺端骨骺损伤的大鼠动物模型,研究骨桥形成的分子病理机制.通过Alcian blue染色观察损伤模型的建立、损伤愈合过程以及骨桥形成情况.采用Tunel试剂盒原位细胞凋亡检测,了解损伤区及周围细胞凋亡情况.利用免疫组织化学及原位杂交实验,观察损伤区周围软骨细胞改变,检测损伤区是否有软骨细胞生成,检测Ihh以及Ptch1表达阳性细胞.发现骨骺损伤骨桥形成过程中,完全损伤区中心没有软骨细胞特异的因子Col2a1和ColⅩ以及Ihh和Ptch1的表达,但是完全损伤区和周围正常软骨交界间存在次损伤软骨区,存在软骨细胞凋亡,有ColⅩ的表达,Vimentin检测发现,在此区和周围正常软骨间有正常肥大区软骨细胞异常分化而来的成纤维样细胞并形成软骨外膜样结构,次损伤区和软骨外膜结构逐渐被骨桥替代,在此过程中软骨外膜样结构存在Col1a1、Ptch1和Ihh的表达,提示Ihh可能参与骨桥形成过程.提出骨桥形成过程中损伤中心区域存在膜内化骨,边缘区域存在软骨化骨作用机制.

Children Salter＇ s type Ⅲ and Ⅳ growth plate injuries always induce the skeletal deformity because of bony bridge formation. However, the underlying cellular and molecular changes of the remaining cartilage adjacent to the injury site are still unclear. The purpose of this investigation was to understand the molecular mechanisms of bony bridge formation. The in vivo cellular and molecular changes in the adjacent cartilage were studied in the rat growth plate injury models. Consisted with the histological changes, both Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling （TUNEL） assay and in situ hybridization experiment using Col2al probe showed there was the sub-injury cartilage region adjacent to the original injury site. Despite the sub-injury region remained normal cartilage structure and Collagen type X in the extracellular matrix, the chondrocytes within this region showed the dislocation with the cartilage lacunas, and the strand breaks of cleaved DNA in TUNEL assay. That these chondrocytes didn＇t express Col2al mRNA further confirmed they were dead cells. Along with the degradation of sub-injury cartilage, some fibroblast-like cells presented to the cartilaginous region between the sub-injury region and uninjured cartilage. In situ hybridization experiment for Patched 1 （Ptchl）, indicator of Indian Hedgehog （IHH） signaling, indicated these fibroblast-like cells could respond to Hh signaling. These results suggest that the bony bridge formation involves series of changes of chondrocytes and Ihh signaling may be involved in the formation of the transient perichondrium-like structure between the sub-injury cartilage and normal cartilage, and partially contribute to the bony bridge formation. Investigating the underlying cellular and molecular changes after the transphyseal injury will contribute us to explore a prevention treatment in the future clinic.