Komatsu D E, Hadjiargyrou M
Department of Biomedical Engineering, Stony Brook University, NY 11794-2580, USA.
Bone. 2004 Apr;34(4):680-8. doi: 10.1016/j.bone.2003.12.024.
One of the immediate sequelae of bone fracture is regional hypoxia resulting from vasculature disruption. Hypoxia stabilizes and activates the transcription factor hypoxia inducible factor-1alpha (HIF-1alpha), which ultimately leads to HIF-1-regulated gene expression. Because nothing is known about HIF-1 involvement in bone regeneration, we performed a series of experiments to elucidate the expression pattern of HIF-1alpha and selected HIF-1 target genes using a rat femoral fracture model. Callus samples were obtained on postfracture days (PFD) 3, 5, 7, 10, 14, and 21. Quantitative RT-PCR (qRT-PCR) was employed to quantify the temporal mRNA expression patterns of HIF-1alpha, vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and heme oxygenase-1 (HO-1). Elevated HIF-1alpha and VEGF expression was seen at all time points, with peak increases of approximately 6- and 2-fold relative to the intact bone present on PFD 10 for HIF-1alpha and VEGF, respectively. Robust activation of iNOS was detected solely on PFD 10 (6.8-fold) with all other time points showing slight downregulation. HO-1 expression peaked on PFD 3 (4.5-fold) with no significant changes on any other PFD. Western blot analysis verified the temporal expression patterns with HIF-1alpha protein expression showing a steady rise to a PFD 10 peak of approximately 18-fold. Similarly, the expression patterns for VEGF and HO-1 showed increases of approximately 4-fold at their PFD 10 and PFD 3 peaks, respectively. Immunohistochemical analysis of PFD 10 callus sections revealed coexpression of HIF-1alpha and VEGF in proliferating chondrocytes and active osteoblasts. Immunostaining for HO-1 on PFD 3 callus sections demonstrated strong expression in hematoma macrophages and vascular endothelial cells. Taken together, these experiments demonstrate for the first time that HIF-1alpha is upregulated at both transcriptional and translational levels in the fracture callus and indicate that PFD 10 may be a key angiogenic time point in the developing rat fracture callus.
骨折的直接后遗症之一是因血管系统破坏导致的局部缺氧。缺氧会使转录因子缺氧诱导因子-1α（HIF-1α）稳定并激活，最终导致HIF-1调控的基因表达。由于对HIF-1参与骨再生的情况尚不清楚，我们利用大鼠股骨骨折模型进行了一系列实验，以阐明HIF-1α的表达模式并筛选HIF-1靶基因。在骨折后第3、5、7、10、14和21天获取骨痂样本。采用定量逆转录聚合酶链反应（qRT-PCR）来定量分析HIF-1α、血管内皮生长因子（VEGF）、诱导型一氧化氮合酶（iNOS）和血红素加氧酶-1（HO-1）的时间性mRNA表达模式。在所有时间点均观察到HIF-1α和VEGF表达升高，相对于骨折后第1天完整骨，HIF-1α和VEGF在骨折后第10天的峰值分别增加了约6倍和2倍。仅在骨折后第10天检测到iNOS的强烈激活（6.8倍），其他所有时间点均显示轻微下调。HO-1表达在骨折后第3天达到峰值（4.5倍），在其他任何骨折后天数均无显著变化。蛋白质印迹分析证实了时间性表达模式，HIF-1α蛋白表达稳步上升，在骨折后第10天达到峰值，约为18倍。同样，VEGF和HO-1的表达模式在其骨折后第10天和第3天的峰值分别增加了约4倍。对骨折后第10天骨痂切片的免疫组织化学分析显示，HIF-1α和VEGF在增殖的软骨细胞和活跃的成骨细胞中共表达。对骨折后第3天骨痂切片进行HO-1免疫染色显示，在血肿巨噬细胞和血管内皮细胞中有强表达。综上所述，这些实验首次证明骨折骨痂中HIF-1α在转录和翻译水平均上调，并表明骨折后第10天可能是发育中的大鼠骨折骨痂血管生成的关键时间点。
