Wnt/β-catenin信号通路在脑脊液搏动调控新生椎板成骨与塑形中的作用机制研究

Dural adhesion and loss of spinal stability postlaminectomy have been urgent problems in clinic. In previous work, we demonstrated that physiological lamina reconstruction could be achieved with tissue engineering technology, and found that the newborn lamina with the regulation of cerebrospinal fluid (CSF) pulsation gradually remodeled to an arched structure that well matched the spinal dural sac. However, the mechanism of the CSF pulsation regulatory function is unclear. Combining with the previous result that the higher expression of Wnt3a in the CSF pulsation unsegregated group, we speculated that canonical Wnt/β-catenin signaling pathway played an important role in the CSF pulsation regulating newborn lamina osteogenisis and remodeling. To confirmed our speculation, we will built CSF pulsation segregated and unsegregated animal model in vivo, and compare the expression of ERK1/2, Akt, Wnt3a, BMP-2, OPG, RANKL and the volum and arrangement of trabecular bone to verify that the canonical Wnt/β-catenin signaling pathway could be activated by CSF pulsation with the transduction of mechanical signals to biochemical signals, which would subsequently regulate the newborn lamina osteogenisis and remodeling. As a parallel experiment, we will built CSF pulsation simulating mechanical model in vitro to verify whether the canonical Wnt/β-catenin signaling pathway was activated by CSF pulsation. And the effect of the canonical Wnt/β-catenin signaling pathway activated by CSF pulsation on osteoblasts proliferation , differentiation, migration and the underlying mechanism will be investigated via comparing the expression of Wnt3a, β-catenin, ALP, OCN and OPG under the condition that the osteoblasts are affected with or without CSF pulsation, and that the signaling pathway is reactivated or inhibited. This investigation will provide theoretical basis to the clinic application of the bone tissue engineering vertebral lamina reconstruction technology.

椎板切除术后硬膜粘连、脊柱失稳是临床亟待解决的难题。前期研究证实组织工程可生理性重建椎板，脑脊液搏动调控椎板形成与硬膜囊匹配的拱形结构，但机制尚不明确。预实验发现Wnt3a在脑脊液搏动未隔离组高表达，推测：Wnt/β-catenin信号通路在脑脊液搏动调控新生椎板成骨与塑形中发挥重要作用。鉴于此，拟构建脑脊液搏动隔离和未隔离动物模型，对比ERK1/2、Akt、Wnt3a、BMP-2、OPG、RANKL的表达及骨小梁体积、排列方向，体内验证脑脊液搏动通过ERK1/2、Akt通路将力学信号转化为生化信号，激活Wnt/β-catenin通路调控椎板成骨与塑形；构建脑脊液搏动力学模型，对比该通路被脑脊液搏动激活、过表达或抑制情况下Wnt3a、β-catenin、ALP、OCN、OPG的表达，体外研究该通路在脑脊液搏动调控成骨细胞增殖分化迁移中的作用及机制；为组织工程重建椎板的临床应用提供理论基础。

椎板切除术是脊柱外科常用术式，用于治疗肿瘤、创伤以及退行性病变等脊柱疾病，但术后脊柱不稳、硬膜粘连等并发症仍是国际性难题。近年来，临床上尝试了多种方式重建椎板，但效果都不理想。项目组利用组织工程技术重建椎板，证实组织工程技术在解决该难题的独特优势。同时，项目组发现脑脊液搏动能够促进组织工程椎板的成骨以及塑形，但具体调控机制尚不可知。查阅文献以及预实验提示脑脊液搏动可能通过Wnt/β-Catenin信号通路调控组织工程椎板的形成。本研究拟通过体内以及体外试验探讨脑脊液搏动通过Wnt/β-Catenin信号通路调控组织工程椎板成骨和塑形的作用机制。在本研究中，项目组检测了多种动物腰椎段脑脊液压力参数以及椎管参数，利用有限元分析构建脊椎-脑脊液力学模型，设计研发了脑脊液搏动生物反应器。借助生物反应器开展体外细胞实验，证实了脑脊液搏动能通过Wnt/β-catenin信号通路来促进间充质干细胞的成骨分化，促进血管生成因子的表达，而这与PI3K/Akt、ERK1/2信号通路无直接关系，脑脊液搏动对间充质干细胞的增殖无显著影响。同时，项目组构建了脑脊搏动动物模型、脑脊液隔离动物模型以及脑脊液搏动+β-Catenin抑制动物模型，发现脑脊液搏动组组织工程椎板的血管化、成骨和骨重塑指标显著优于脑脊液隔离组和脑脊液搏动+β-Catenin抑制组，验证了脑脊液搏动通过激活Wnt/β-catenin信号通路来促进组织工程椎板的血管化、成骨和塑形。并且初步发现脑脊液搏动可能是通过YAP蛋白来实现力学-生物学信号转化，从而激活Wnt/β-catenin信号通路，具体作用机制需要进一步探讨。综上所述，本项目通过体内以及体外试验验证了脑脊液搏动通过Wnt/β-catenin信号通路促进间充质干细胞的成骨分化，进而促进组织工程椎板的血管化、成骨和塑形，为组织工程重建椎板技术的临床转化应用提供了理论基础。