GSK3β/β-catenin信号轴耦合成骨和血管形成在上颌骨缝改建中的作用

Mechanical strain across craniofacial sutures is well recognized as a controlling stimulus for bone formation and remodeling in the skull and face. This is the basis upon which growth modification of the maxillary complex can be achieved through various orthopedic-orthodontic appliances. Recent researches suggested that GSK3β/β-catenin signaling is a normal physiological response to mechanical loading in bone. This signaling pathway mediates skeletogenesis by promoting osteoblast proliferation and function. Moreover, it promotes angiogenesis through activating the renewal of endothelial cells. Our previous works showed that elevating β-catenin expression by GSK3β inhibition enhanced bone deposition in expanding maxillary suture in rat. Thus, the present research proposal is designed to test if manipulation of GSK3β/β-catenin signaling could enhance the growth modification of maxillary sutures upon orthodontic forces. A co-culture of rat calvarial ostoblasts and vascular endothelial cells will be subjected to mechanical stretches by Flexcell 4000 strain unit. In parallel, maxillary suture expansion in rat will be carried out. The signaling inhibitors and mutants of both constitutively active form and dominant negative form of GSK3β will be delivered to the in vitro and in vivo experiments. The proliferation, differentiation and function of osteoblasts and endothelial cells will be assessed by histomorphometry, immnohistochemistry and PCR. In this way, the functions of GSK3β/β-catenin axis in integrating angiogenesis and osteogenesis in maxillary sutures upon mechanical loading will be clarified.

上颌骨缝在机械力作用下的生长改建是正畸治疗颌面部畸形的基础。GSK3β /β-catenin信号轴是骨组织应答机械应力的生理反应途径，可以促进成骨细胞的形成，同时促进血管新生。我们前期的研究表明，使用GSK3β抑制剂可以促进β-catenin的表达，进而促进前腭缝的牵张成骨。本研究拟通过成骨细胞和血管内皮细胞应力加载以及大鼠上颌骨缝牵张模型，使用通道抑制剂和基因修饰的方法干预GSK3β /β-catenin信号通路，应用组织测量学、免疫组织化学、PCR等技术，研究上颌骨缝生长改建过程中GSK3β /β-catenin信号分子的表达及其作用，研究牵张力促进血管形成和促进成骨双重效应的机制及其与GSK3β/β-catenin信号的关系，研究GSK3β/β-catenin信号调控促进骨缝牵张成骨的效果，以此阐明正畸力作用下上颌骨缝生长改建的分子生物学机理，进而探索生物干预促进正畸治疗的方法。

应力刺激和血管新生在上颌骨缝生长改建中发挥重要作用，GSK3β/β-catenin信号是骨组织应答机械应力的生理反应途径，同时参与血管的形成。本研究通过大鼠动物实验，发现锂盐作为GSK3β抑制剂可以促进拔牙创β-catenin表达，术前给药促进前成骨细胞的增殖，术后给药促进成骨细胞的分化，两者均明显增加新骨的形成。大鼠前腭缝扩张实验显示，牵张力诱导骨缝内成骨细胞和血管内皮细胞表达β-catenin，锂盐通过促进β-catenin上调VEGF的表达，促进新生血管的形成，招募更多的前成骨细胞，最终促进上颌骨缝扩张成骨。体外实验显示，成骨细胞和血管内皮细胞以2:1的比例直接共培养时，对成骨基因Runx2、OCN和成血管基因vWF、ET-1表达的协同促进作用最明显，并紧随着VEGF表达的显著提高。对共培养细胞使用Flexcell 4000施加6%等轴循环牵张力12h，诱导β-catenin的一过性增加，进而促进细胞的增殖，这主要是牵张力诱导内皮细胞表达β-catenin的结果。并且牵张力和LiCl诱导的β-catenin对细胞增殖有协同促进作用。这种短暂的β-catenin增加伴随着随后共培养细胞Runx2和ALP表达的增加。这些体内外实验显示，GSK3β/β-catenin信号是促进颌骨生长和改建的一个重要靶点；β-catenin对成骨细胞的增殖和分化有双向调控作用；牵张力通过诱导内皮细胞表达β-catenin而促进成骨细胞的活性，是机械应力促进成骨的又一重要途径。这对临床探索促进上颌骨生长改良的方法，以及应用组织工程等手段促进颌骨缺损修复有重要参考价值。但内皮细胞表达β-catenin促进成骨细胞功能的机制尚需进一步阐明。