HIF-Wnt在低氧微环境下协同调控老龄BMSCs成骨的机制研究

Aged patients have osteoporosis and poor healing ability after comminuted jaw bone fracture. Non-union is one of the serious post-operation complications. Local hypoxia microenvironment caused by comminuted fracture and its influence on bone marrow derived mesenchymal stem cells (BMSCs) play key role in the procedure of fracture healing. BMSCs might be injured or show chronic adaptation to hypoxia condition depends on how serious it is and what kind of treatment it get. Therefore, how to improve these cell to fit to hypoxia circumstance and promote their osteogenic capacity is one of the most important issue to accelerate the comminuted bone fracture healing process in the old patients. Hypoxia induced fator (HIF) is one of the good factors that improve the vitality of BMSCs. However, HIF might also be regulated by other osteogenesis related signaling pathways. We have demonstrated that Wnt3a had the ability to reverse the senile status of aged BMSCs and promote their bone formation ability. Previous studies of other diseases showed that Wnt could have crosstalk with HIF in hypoxia environment. These results indicate that the cooperation of HIF and Wnt has the possibility of promoting bone healing in hypoxia condition. Based on our previous studies, we plan to employ various cellular and molecular methods to explore the characteristics of BMSCs under hypoxia and the molecular mechanism of HIF-Wnt crosstalk on the regulation of BMSCs osteogenesis. This research will be highly valued in promoting bone formation capacity in aged BMSCs and its application in the comminuted jaw bone fractures in osteoporosis individuals. Besides, this study would also meaningful to the management of osteoporosis related diseases and comminuted fracture beyond oral-maxillofacial bones.

老年颌骨伴骨质疏松更易发生粉碎性骨折，术后也更多出现骨不连等棘手问题，粉碎骨折局部特有缺血低氧微环境可能在其中发挥了关键影响。骨髓间充质干细胞(BMSCs)参与骨折修复时，在低氧微环境下既有损伤，若调控有力亦可呈现慢性适应，如何促进细胞适应低氧、增强成骨，是改善老龄个体粉碎骨折愈合的一个关键问题。低氧诱导因子（HIF）的升高是改善BMSCs活性的有益因素，但HIF也可能受控于成骨信号通路，既往研究显示Wnt在低氧下与HIF可能有交互，提示HIF-Wnt协同作用有望作为改善低氧下成骨的重要手段。本课题基于前期基础，拟采用多种细胞学和分子学手段，探讨低氧下BMSCs特性，以及HIF-Wnt协同调控BMSCs成骨的分子机制。该研究对于提升低氧下老龄BMSCs成骨，并促进其在颌面骨质疏松粉碎性骨折愈合中的应用有重要意义，对于其它领域如骨质疏松相关疾病干预、身体其它部位粉碎骨折研究亦有参考价值。

目前中国社会老龄化趋势严峻，各方面压力都越来越大。老年疾病治疗中亟待解决的难题也越来越多。老年粉碎性骨折的愈合就是难题之一，因为局部血供遭到破坏，处于缺血低氧状态，不利于骨再生。本课题研究骨损伤愈合过程中，局部低氧微环境的负面影响以及如何改善低氧下BMSCs再生骨骼的能力。包括：低氧微环境中老龄BMSCs 生物学行为的改变，低氧预处理及相关信号对BMSCs生存、成骨、成血管的影响，低氧预处理对BMSCs体内成骨能力及相关信号分子的影响等内容。研究结果发现：老龄BMSCs表面抗体流式鉴定，符合成体间充质干细胞特点；老龄个体BMSCs成骨潜能较年轻个体减弱，并且与低氧相关；细胞增殖能力在初始接触低氧环境后都会出现增殖能力下降的趋势，而后都会逐渐上升；发现1%氧+无血清的环境比21%氧+10% FBS出现更多的死亡，提示高氧培养的细胞转移到到低氧+缺血的体内微环境中，可能有大量损失；将BMSCs预先进行低氧处理，再置入1%氧+无血清区域，则发现死亡数将大大减少；我们还发现BMSCs形成三维球体，在缺血缺氧的条件下，比零散的BMSCs表现出更强的活性；HIF保护剂DMOG+低氧预处理对于提高细胞移植存活率具有双重正性作用；低氧预处理可提高BMSCs中HIF-1α及VEGF的蛋白表达量，而低氧预处理辅以DMOG的双重作用，则在成管实验中形成了更多的管型；通过多次分组，发现低氧+DMOG对细胞的成骨潜力具有积极的刺激作用；建立老龄鼠下颌骨缺损模型，不同处理方法的细胞与生物材料复合后，植入骨缺损区域，在不同时间点对标本进行影像学、组织学、细胞学的观察，ALP和ARS染色检测结果表明经低氧预处理的BMSCs在在低氧环境下具有较强的成骨能力，Micro-CT，HE和Masson染色以及免疫组织化学染色表明经低氧预处理的BMSCs在体内具有较强的修复骨缺损能力。综上，本研究进一步阐述了传统高氧培养给细胞带来的弊端，证明了低氧处理对细胞生物学行为的改变及提高细胞在恶劣环境生存的能力，以及相关信号因子在其中发挥的调控作用，这为提高细胞从体外移植到体内进行组织器官再生的效果，提供了实验依据；同时，本研究针对老龄细胞，为解决老龄个体骨修复过程中存在的难题提供了新的线索。