HIF-1α通过耦联调控H型血管内皮发生和骨形成促进骨再生的作用机制研究

Bone defect healing is a common clinic issue. Vascularization of bone graft is a key issue to determine the final performance of tissue-engineered bone and is a key scientific problems puzzled for bone regeneration. Consistence between bone development and regeneration give us a cue to seek solution from the coupling of angiogenesis and osteogenesis during the bone development and repair healing, but the underlying molecular mechanisms responsible for the cross-talk between angiogenesis and osteogenesis are not well defined. Previously, we found that HIF-1α is a key transcriptional factor for the coupling between angiogenesis and osteogenesis. Most recently, it was reported that there is a new founded type H endothelium cells in the bone marrow, which is highly related with expression of HIF-1α and play an important role in angiogenesis and bone formation. Therefore, we propose a new cross-talking molecular mechanism by which HIF-1α regulate the coupling of type H endothelium and osteogenesis. This project aims to explore the mechanism of HIF-1α in the coupling of angiogenesis and osteogenesis by means of MACS and FACS-mediated cell sorting, lentivirus-mediated HIF-1α overexpression and knock down, direct and indirect cell co-culture, and Matrigel tube formation approaches. Based on understanding the underlying mechanism, we will target HIF-1α with hypoxia mimic agent deferoxamine (DFO), improve our previously developed HC-ACBM scaffold using electrostatic self-assembly methods, and construct prevascularized tissue-engineered bone by co-seeding with H-ECs and MSCs. Then we will evaluate the ectopic bone formation and neo-angiogenesis of this prevascularized tissue-engineered bone by in vivo transplantation and the stimulating effects on the regeneration and reconstruction of bone defects using rabbit diaphyseal defects model. Our study will uncover the molecular mechanism responsible for the coupling of angiogenesis and osteogenesis, and develop a new strategy promoting bone defects healing based on our new finding.

骨缺损是临床常见问题。血管化问题限制了组织工程骨的最终治疗效果。组织发育和再生的相似性提示可以从血管新生藕联骨生成中寻找对策，目前两者“串话”的分子机制还不清楚。我们前期发现低氧诱导因子HIF-1α在藕联血管新生和骨生成中至关重要。最新报道骨髓中存在一种H型血管内皮细胞，与HIF-1α表达密切相关，影响血管生成和骨形成。因此，我们提出HIF-1α调节H型血管内皮发生藕联骨生成的机制假说。本课题我们将利用细胞分选、慢病毒感染、细胞共培养、Matrigel小管形成实验等方法阐明HIF-1α藕联血管新生与骨生成的分子机制。在此基础上以HIF-1α为靶点，利用静电自组装技术改进前期研制的HC-ACBM支架材料，并以H-ECs和MSCs为种子细胞构建预血管化组织工程骨，考查异位成骨和血管生成情况以及对骨缺损修复的促进作用。我们的研究将揭示血管新生与骨生成的藕联机制，并据此提出促进骨缺损再生的新策略。

骨骼是一个高度血管化的组织，血管新生（angiogenesis）是骨骼发育和再生修复的一个基本环节。研究发现低氧诱导因子HIF-1α在耦联血管新生和骨生成中发挥关键作用。瞄准血管新生耦合骨生成的生物学现象，以HIF-1α为作用靶标，在促进骨缺损修复中有良好的应用前景。通过本研究，明确了靶向HIF-1α的不同浓度DMOG（0, 50μM, 100μM）对前成骨细胞系MC3T3-E1的细胞增殖、成骨分化及VEGF表达的影响，发现常氧条件下低氧模拟剂DMOG明显促进成骨细胞增殖和VEGF表达，而抑制MC3T3-E1的成骨分化。明确了靶向HIF-1α的低氧模拟剂DFO对BMSCs在增殖、克隆形成、成骨和成脂分化、细胞衰老相关分子检测以及对血管生成的作用，证明体内激活HIF-1α对衰老骨骼BMSCs活力的改善作用及分子机制。考察了靶向HIF-1α的低氧模拟剂对血管内皮细胞的成管能力的影响；利用基因芯片系统分析了低氧信号对血管内皮细胞基因表达谱的影响。明确了靶向HIF-1α的低氧模拟剂去铁胺药物DFO通过靶向骨形成和血管形成在治疗大鼠老年性骨质疏松中有显著的作用，我们的研究结果表明，高剂量甲磺酸去铁胺（60mg/kg）对老年大鼠的骨质疏松有明显的治疗作用。去铁胺DFO目前在临床用于治疗多铁症患者，我们的研究结果表明去铁胺在骨质疏松治疗中的新作用。基于HIF-1a在耦连血管生成与骨再生中的关键作用，我们制备了构建了缓释DMOG的新型煅烧骨支架材料DMOG/COL I/TBC，明确了该复合支架材料的理化性质，包括形貌特征、物相组分、释放特征、细胞相容性，发现该新型支架材料与BMSC细胞有很好的相容性，能促进BMSC表达成骨和成血管的关键基因，DMOG负载至COL I/TBC支架材料实现了一周的缓慢释放，动物实验表明将DMOG负载到COLI/TBC支架材料能促进兔股骨髁骨缺损的修复。我们的研究表明荷载低氧模拟剂DMOG可以改进煅烧骨的骨诱导活性，为骨缺损修复及植骨材料的研制提供了新的思路。