Hippo信号通路节点YAP/TAZ介导种植体骨结合中免疫响应的作用及机制研究

There is a great demand for dental implant restoration in patients. Clinically, it is faced with long healing time, poor osseointegration and other challenges. The immune response performed by macrophages is critical to the early formation and long-term stability of the implant-bone interface. However, the intrinsic transduction mechanism of the immune response associated with osseointegration has not been elucidated. Our previous studies indicated that Hippo signaling effectors YAP/TAZ is closely related to both macrophages M1/M2 polarization and implant osseointegration affected by neuropeptides, suggesting that the Hippo pathway is expected to mediate immune response and osteogenesis through its effectors YAP/TAZ. Therefore, in the view of macrophages immune response, we combine gene knockout and overexpression technologies to obtain related target genes differential expressions in macrophages. In vitro study of Hippo-YAP signaling-mediated interaction between macrophages and mesenchymal stem cells (MSCs) will be explored first, combined with CRISPR/Cas9 technique for reverse validation. Furthermore, animal models with titanium implants will be established to evaluate the role and underlying mechanism of Hippo signaling effectors YAP/TAZ in triggering immune response and cascade regulation of bone regeneration during osseointegration. Live fluorescence tracing, histomorphology analysis and various gene and protein detection techniques will be conducted in these animal studies. The results of this program will be of great significance in achieving rapid and stable implant osseointegration, as well as the improvement of implant restoration in patients.

牙种植社会需求大，临床上面临愈合时间长、骨结合不良等挑战。巨噬细胞执行的免疫响应对种植体骨结合界面的早期形成和长期稳定至关重要，但有关免疫响应等骨结合系列事件的内在转导机制尚未阐明。本课题组前期发现，Hippo信号通路效应蛋白YAP/TAZ与神经肽影响下的巨噬细胞M1/M2极化及种植体骨结合相关，提示Hippo通路有望通过YAP/TAZ节点介导免疫响应及成骨效应。基于此，本课题以巨噬细胞的免疫响应为切入点，通过基因敲除及过表达获得巨噬细胞内关键基因的表达差异，体外探究Hippo-YAP信号介导下巨噬细胞和干细胞的交互作用，同时结合CRISPR/Cas9技术反向验证；体内构建钛种植体植入模型，采用活体荧光示踪、组织形态学分析及基因蛋白检测等，评价YAP/TAZ节点触发骨结合中免疫响应、级联调控骨再生的作用及潜在转导机制。研究结果对于实现快速稳定的种植体骨结合、提高种植修复效果，具有重要意义。

牙种植社会需求大，临床上面临愈合时间长、骨结合不良等挑战。巨噬细胞执行的免疫响应对种植体骨结合界面的早期形成和长期稳定至关重要，但有关免疫响应等骨结合系列事件的内在转导机制尚未阐明。本课题组前期发现，Hippo信号通路效应蛋白YAP/TAZ与神经肽影响下的巨噬细胞M1/M2极化及种植体骨结合相关，提示Hippo通路有望通过YAP/TAZ节点介导免疫响应及成骨效应。基于此，本课题以巨噬细胞的免疫响应为切入点，首先体外探究神经肽类物质降钙素基因相关肽（CGRP）对巨噬细胞极化的影响，发现敲除CGRP会促进原代巨噬细胞的M1型极化，抑制M2型极化；在此基础上，深入探究机制，发现CGRP可以通过Hippo-YAP信号参与调节M2型巨噬细胞促修复因子的分泌，并进一步调节成骨前体细胞系MC3T3成骨向分化；此外，本项目验证了Hippo-YAP信号通路体外参与调控骨髓间充质干细胞（BMSCs）成骨；构建BMSCs和内皮细胞（ECs）共培养模型，发现BMSCs能通过Hippo-YAP信号促进ECs的增殖和迁移能力；体内构建小鼠上颌骨种植模型，采用荧光示踪、组织形态学分析及基因蛋白检测等研究CGRP调控巨噬细胞极化介导种植体骨结合的作用，表明CGRP可以通过抑制颌骨种植体周巨噬细胞的M1表型、促进M2表型来促进成骨；同样应用此方法，探究Hippo-YAP信号轴在CGRP介导下调控骨改建进程中成骨成血管的交互效应，发现CGRP可以介导Hippo-YAP信号轴上调种植体周围成骨、成血管表型的表达，促进局部骨愈合。以上研究结果证实了Hippo-YAP信号轴在触发骨结合中免疫响应、血管生成及级联调控骨再生中的重要作用；开辟性地以Hippo-YAP信号轴为切入点，将免疫、骨组织和血管系统结合在一起，提示其可能成为种植体周骨-血管-免疫交联的重要靶点。本项目研究结果对于实现快速稳定的种植体骨结合、提高种植修复效果，具有重要意义。