Institute of Sports Medicine, Peking University Third Hospital, No. 49, North Garden Road, Haidian District, Beijing 100191, People's Republic of China.
Bone. 2010 Feb;46(2):425-31. doi: 10.1016/j.bone.2009.08.057. Epub 2009 Sep 6.
Animal model for heterotopic ossification (HO) induced by Achilles tenotomy in rats has been used in the literature. However, the molecular mechanism remains unclear. Here, we studied bone and cartilage related genes and their possible roles in this model. Thirty rats underwent bilateral midpoint Achilles tenotomy through a posterior approach under aseptic condition. At 3, 5 and 10 weeks post-operation, X-ray and histological examinations were carried out to investigate the formation of HO. At different phases of HO formation, mRNA levels of transforming growth factor (TGF)-beta1, TGF-beta2, TGF-beta 3, bone morphogenetic proteins (BMP)-2, BMP-4, BMP-7, hypoxia inducible factor (HIF)-1 alpha, Sox9, Runx2, vascular endothelial growth factor (VEGF), aggrecan and collagen type I, II and X were evaluated by real-time RT-PCR. Protein levels of TGF-beta1, TGF-beta2, TGF-beta 3, BMP-2, BMP-4, BMP-7, HIF-1 alpha, Sox9 and Runx2 were also examined by immunohistochemical staining. During the chondrogenic phase, the expressions of HIF-1 alpha and Sox9 were significantly up-regulated. Runx2 expression was significantly up-regulated during osteogenic phase, while HIF-1 alpha and Sox9 expression was significant decreased. TGF-beta1 mRNA levels were rather constant, and the mRNA levels of TGF-beta2, TGF-beta 3 and BMPs were changed throughout HO formation. The presences of the proteins of HIF-1 alpha, Sox9, Runx2, TGF-betas and BMPs within the HO tissues were confirmed by immunohistochemical staining. Our study indicates that HO induced by Achilles tenotomy is by endochondral bone formation, and HIF-1 alpha activation plays an important role during chondrogenesis in this model. Furthermore, the model provides a new experimental system to study endochondral ossification.
在文献中，已经使用了通过跟腱切断术在大鼠中诱导异位骨化（HO）的动物模型。然而，其分子机制尚不清楚。在这里，我们研究了骨和软骨相关基因及其在该模型中的可能作用。30 只大鼠在无菌条件下通过后路进行双侧跟腱中部切断术。在术后 3、5 和 10 周时，进行 X 射线和组织学检查以研究 HO 的形成。在 HO 形成的不同阶段，通过实时 RT-PCR 评估转化生长因子（TGF）-β1、TGF-β2、TGF-β3、骨形态发生蛋白（BMP）-2、BMP-4、BMP-7、缺氧诱导因子（HIF）-1α、Sox9、Runx2、血管内皮生长因子（VEGF）、聚集蛋白聚糖和 I、II 和 X 型胶原的 mRNA 水平。通过免疫组织化学染色还检查了 TGF-β1、TGF-β2、TGF-β3、BMP-2、BMP-4、BMP-7、HIF-1α、Sox9 和 Runx2 的蛋白水平。在软骨形成阶段，HIF-1α和 Sox9 的表达明显上调。在成骨阶段，Runx2 表达明显上调，而 HIF-1α和 Sox9 的表达明显下调。TGF-β1 的 mRNA 水平相对恒定，而 TGF-β2、TGF-β3 和 BMPs 的 mRNA 水平在 HO 形成过程中发生变化。通过免疫组织化学染色证实了 HO 组织中 HIF-1α、Sox9、Runx2、TGF-βs 和 BMPs 蛋白的存在。我们的研究表明，通过跟腱切断术诱导的 HO 是通过软骨内骨形成，并且在该模型中的软骨形成过程中 HIF-1α 激活发挥重要作用。此外，该模型为研究软骨内骨化提供了新的实验系统。
