钛表面硅烷化修饰GL13K对巨噬细胞极化状态的影响研究

With the arising of the osteoimmunology, many study had confirmed that the bone metabolism is close relatived with body immunity and positive feedback of immue is good for bone regeneration. Macrophage is the major target cell in phase of immune process and the polarization of macrophage is also important to the bone regeneration. Therefore, how does the modification of the implant surface influence the macrophage in the process of immune is still need further research. The antibacterial peptide GL13K was successfully immobilized onto the titanium surfaces which performed antimicrobial activity well and down-regulated the pro-inflammatory cytokines in our early experiment. Downgrading the inflammatory reaction after implant placement and then promoting osteoimmunology activity transform to the bone regeneration and repair process, using the macrophage as a target cell, are the aim of our study. Firstly, Liquid Protein Chip, qRT-PCR and FCM will be used to test secretion of cytokines, the macroghage phenotype of M1/M2 and the possible signal pathway of how the antimicrobial peptides GL13K coating affect the secretion of cytokines. Sceondly, TRAP staining, co-culture and qRT-PCR was used to measured the osteogenesis, osteoclastogenesis and angiogenesis of the BMSCs and HUVECs in the macrophage condition medial, and the influence of the BMP-2, M-CSF and VEGF on it by adding correspondenting receptor antagonist. Our goal is to reducing the inflammation reaction after implant placement and then speed up the tissue regeneration by studying how the polarization of macrophage and the relatively cytokines influence the osteogenesis, osteoclastogenesis and angiogenesis, promoting osteoimmunology activity transform to the bone regeneration, laying the foundation of how the modification of implant surface influence the immuno-environment, and providing a new ideas for the implant design.

随着骨免疫概念的提出，研究发现骨代谢与机体免疫调节关系密切，正向的免疫反馈有利于骨组织再生，巨噬细胞是免疫应答的主要宿主调节细胞，其M1/M2极化状态的转变对组织代谢发挥重要作用。因此，种植体表面改性后如何影响巨噬细胞在免疫应答中的作用有待深入研究。我们前期实验已将抗菌肽GL13K修饰到钛表面，并发挥良好抗菌性能和下调巨噬细胞分泌促炎因子。本课题从种植体植入后的免疫反应入手，以巨噬细胞为核心，通过液相蛋白芯片、qRT-PCR、流式细胞术等手段检测细胞因子分泌、极化状态及信号通路；通过共培养、qRT-PCR等研究巨噬细胞对成骨、破骨和血管再生方面的影响，应用相关受体拮抗剂研究BMP-2/M-CSF/VEGF影响途径。通过研究巨噬细胞极化状态及分泌相关细胞因子如何影响骨免疫中成骨、破骨和血管再生，进一步探讨钛表面改性后免疫调节促进骨再生转归的分子机制。

近年来越来越多研究致力于钛材料表面改性，过各种材料表面改性的技术赋予其表面新的优良性能，以获得更牢固、稳定的种植体-骨结合率，提高种植成功率。本课题以巨噬细胞为主要研究对象、种植体表面免疫反应为切入点，深入研究巨噬细胞极化状态的转变及相应细胞因子的分泌对免疫相关成骨再生、成血管再生的影响。首先我们研究了抗菌肽GL13K涂层后钛材料可维持良好的内部结构稳定性，抗电化学腐蚀性能更佳；其次，抗菌肽GL13K涂层钛对骨髓间充质细胞和巨噬细胞均表现出良好的生物相容性，有促进骨髓间充质干细胞相互聚集和适当调节巨噬细胞极化的趋势，进而促进一系列有利于组织修复的细胞生物学行为发生；最后，GL13K涂层钛表面在体外有利于巨噬细胞形成促进组织修复的局部微环境，可适当抑制破骨分化、促进成骨分化和成血管分化。GL13K涂层钛促使种植体-骨界面的组织反应向新骨形成方向发展，从免疫调节促进其向组织愈合方向转归这一全新的角度出发，初步探索了抗菌肽GL13K涂层钛未来在口腔种植材料方面的潜能，并为该材料后续研究奠定了基础，提供了思路。

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