Wnt/β-catenin信号通路在经缝牵引成骨中的作用机制

Facial sutures are fibrous or bony tissue articulations that connect the bones of the midfacial skeleton together and serve as growth sites to stimulate bone formation and regulate the bone remodeling in mechanical environment during childhood. The technique of trans-suture distraction osteogenesis promotes skeleton growth by applying mechanical tensile force to the suture and has been successfully used in clinical for treatment of midfacial hypoplasia. Unfortunately, this treatment are still accompanied by long duration of distraction and essential of using facemask with elastic traction to prevent relapse for some patients. Therefore, how to deal with these problems by enhancing the new bone formation to prevent relapse and shorten the distraction has become the focus. However, the cellular mechanism that regulate the suture osteogenic response to mechanical loading are still unkown. Multiple studies have demonstrated the Wnt signaling pathway is essential for many developmental processes including skeletogenesis but rarely reported to play an role in trans-suture distraction osteogenesis. The canonical Wnt signaling is initiated by releasing Wnt proteins bind to their transmembrane receptors including Lrp5/6 and frizzled, which eventually leads to increased β-catenin levels and translacated into the nucleus to modulate expression of Wnt target genes. Meanwhile，our preliminary test showed a stably expression of several components of Wnt signaling cascade such as Lrp5,Lrp6 and β-catenin in zygomaticomaxillary suture. The RNA-seq results of changes in the zygomaticomaxillary suture between 4Weeks and 3Months old rat has also revealed the canonical Wnt signaling involved in the physiological developmental process. Therefore, we hypothesize that the canonical Wnt signaling has played an critical role in the trans-suture distraction osteogenesis and stimulate new bone formation. In this study, we found that mechanical distraction and stress model in vivo of the facial sutures (zygomaticomaxillary suture) in rat. Further analyses include Micro-CT and imaging study, RNA-seq and qRT-PCR , Histological staining, and molecular cell biology. The aim of this is to clarify the role of Wnt/β-catenin signaling pathway in regulating new bone formation in trans-suture distraction osteogenesis, and further explore the effect of only using agonist of Wnt signaling, or used in combination with mechanical distraction on the osteogenic response of the sutures. Eventually, it is possible for designing small molecular drug acting on the target gene to mimic the effect of mechanical stimuli on the osteogenic response of facial sutures so as to prevent and/ or treat some diseases relating to abnormal growing development of facial sutures.

经缝牵引成骨技术已被临床证实是治疗面中部骨骼发育不全的一种有效方法。由于经缝牵引成骨机制尚未阐明，其存在牵引疗程长、需长期佩戴牵引器维持疗效等问题尚未得到解决。Wnt信号在经缝牵引成骨中的作用如何尚未见报道。我们前期研究表明：大鼠颧上颌缝组织中Wnt信号通路基因均有表达。进一步对4w与3月龄颧上颌缝组织转录组测序比较，Wnt信号激活基因表达大多呈上调，而其抑制基因表达有上调、有下调。PCR结果提示牵引2周后有的Wnt信号激活基因表达上调、抑制基因表达下调。本研究拟建立4周龄大鼠颧颌缝机械牵引和压缩实验模型、局部单独使用外源性Wnt信号激动剂或抑制剂模型及联合机械刺激模型，采用影像学、组织细胞学、分子生物学等检测方法，探索Wnt信号通路在大鼠颧颌缝不同处理方式下成骨反应的调节机制。为探索小分子以模拟机械刺激作用于靶基因或配合机械刺激促进缝组织成骨来预防和治疗一些面缝相关性疾病提供实验依据。

面中部骨骼发育不全是临床上常见的一种颅颌面骨骼畸形，尤其多发生于先天性唇腭裂患者。经缝牵引成骨术虽已被证实为矫正面中部骨骼发育不全的有效方法，但对其成骨机制的研究仍有助于该技术的临床优化。本研究第一部分以4周龄生长发育期SD雄性大鼠为实验动物，成功建立经缝牵引成骨干预面中部骨骼生长发育的实验模型。通过对实验大鼠及临床病例环饶面中部骨骼各缝区结构进行影像学扫描分析，结果发现翼上颌缝区和上颌结节区新骨形成在推动面中部骨骼生长前移中发挥最为重要作用，测量数据显示前者增长量占比75%，而后者增长量占比为25%，这表明经缝牵引成骨促进面中部骨骼生长主要依赖翼上颌缝区成骨的继发性移位和上颌结节区生长的原发性移位。第二部分通过对术后不同时间点各组大鼠颧上颌缝组织进行转录组测序分析，结果显示在受力早期牵引组和压缩组差异基因上调均主要富集在细胞外基质受体相互作用、黏着斑、整合素结合、Hippo及Wnt等信号通路上，而在后期有关细胞增殖分化的信号通路在牵引组中显著上调，在压缩组中则显著下调。这表明经缝牵引成骨过程可能为缝区间充质干细胞在机械力刺激下先经细胞外基质-整合素-细胞骨架路径介导信号转导，而后通过Hippo、Wnt等信号通路调控缝区细胞成骨功能。第三部分对Wnt、Hippo信号通路及成骨蛋白相关基因进行PCR检测，结果发现在牵张力刺激下这些基因均呈显著表达上调，且信号通路之间存在交互作用，提示经缝牵引成骨过程中Hippo和Wnt信号通路可能发挥协同作用来调控缝区间充质干细胞向成骨细胞增殖分化，促进新骨形成。第四部分获取颧上颌缝区组织样本进行细胞培养，分离获得缝区间充质干细胞并传代，对其进行诱导分化及流式细胞学分析检测，结果证实缝区间充质干细胞在体外可向骨、软骨及脂肪细胞方向分化，且阳性表达间充质干细胞表面标记物CD29、CD44、CD73和阴性表达造血干细胞表面标记物CD11b、CD45、CD34。通过对缝区间充质干细胞的分离与鉴定，将为经缝牵引成骨的分子机制研究提供新的策略。