Corbett S A, Hukkanen M, Batten J, McCarthy I D, Polak J M, Hughes S P
Department of Orthopaedic and Trauma Surgery, Imperial College School of Medicine, London, UK.
J Bone Joint Surg Br. 1999 May;81(3):531-7. doi: 10.1302/0301-620x.81b3.8852.
Our aim was to investigate whether nitric oxide synthase (NOS) isoforms, responsible for the generation of NO, are expressed during the healing of fractures. To localise the sites of expression compared with those in normal bone we made standardised, stabilised, unilateral tibial fractures in male Wistar rats. Immunostaining was used to determine the precise tissue localisation of the different NOS isoforms. Western blotting was used to assess expression of NOS isoform protein and L-citrulline assays for studies on NOS activity. Control tissue was obtained from both the contralateral uninjured limb and limbs of normal rats. Immunohistochemistry showed increased expression of endothelial NOS (eNOS) to be strongest in the cortical blood vessels and in osteocytes in the early phase of fracture repair. Western blot and image analysis confirmed this initial increase. Significantly elevated calcium-dependent NOS activity was observed at day 1 after fracture. Inducible NOS (iNOS) was localised principally in endosteal osteoblasts and was also seen in chondroblasts especially in the second week of fracture healing. Western blotting showed a reduction in iNOS during the early healing period. Significantly reduced calcium-independent NOS activity was also seen. No neuronal NOS was seen in either fracture or normal tissue. Increased eNOS in bone blood vessels is likely to mediate the increased blood flow recognised during fracture healing. eNOS expression in osteocytes may occur in response to changes in either mechanical or local fluid shear stress. The finding that eNOS is increased and iNOS reduced in early healing of fractures may be important in their successful repair.
我们的目的是研究负责生成一氧化氮的一氧化氮合酶（NOS）同工型在骨折愈合过程中是否表达。为了与正常骨中的表达位点进行定位比较，我们在雄性Wistar大鼠中制作了标准化、稳定的单侧胫骨骨折模型。免疫染色用于确定不同NOS同工型的精确组织定位。蛋白质印迹法用于评估NOS同工型蛋白的表达，L-瓜氨酸测定法用于研究NOS活性。对照组织取自对侧未受伤肢体和正常大鼠的肢体。免疫组织化学显示，在骨折修复早期，内皮型一氧化氮合酶（eNOS）的表达增加，在皮质血管和骨细胞中最强。蛋白质印迹和图像分析证实了这种最初的增加。骨折后第1天观察到钙依赖性NOS活性显著升高。诱导型一氧化氮合酶（iNOS）主要定位于骨内膜成骨细胞，在软骨细胞中也可见，特别是在骨折愈合的第二周。蛋白质印迹显示在愈合早期iNOS减少。还观察到钙非依赖性NOS活性显著降低。在骨折组织和正常组织中均未发现神经元型一氧化氮合酶。骨血管中eNOS的增加可能介导骨折愈合过程中公认的血流增加。骨细胞中eNOS的表达可能是对机械或局部流体剪切应力变化的反应。骨折早期eNOS增加而iNOS减少这一发现可能对其成功修复很重要。
