甲基化转移酶EZH1在巨噬细胞调控骨缺损修复过程中的机制研究

After implanting biological materials in the body, the body immune response may lead to rejection of implant material as foreign body reaction, on the other hand, it also plays a crucial role in bone regeneration. The applicant has been focusing on the effects of macrophage on bone formation stimulated by different biomaterials through polarization. Previously our group has found the different effect of bone formation by several biomaterials in vitro, and the material can promote osteogenesis through regulating macrophage polarization. Moreover, the expression of histone methylation enzyme EZH1 is different stimulated by different biomaterials. EZH1 can not only catalyze H3K27me1 and H3K27me2 due to the SET domain, but also up-regulate the target genes of NF-κB. This project proposed to understand the role of EZH1 in macrophage polarization after biomaterials implanted by the application of macrophages-knockout mice models and EZH1-knockout mice, and verify the target genes and regulation mechanism of EZH1 through such means as co- immunoprecipitation, ChIP and so on. This project combines in vivo and in vitro experiments to explore the role of EZH1 in regulating bone formation effects of biomaterials through multiple perspectives, enable us to avoid immune-related complications better after biomaterial implantation, as well as to improve the effects and safety of biomaterials.

生物材料植入机体后作为一种异物，体内免疫反应一方面可能导致植入材料的排斥，另一方面在有效的骨再生中也起到至关重要的作用。申请人聚焦在巨噬细胞通过向不同亚型分化从而影响材料成骨效果的作用。前期发现不同材料在体内体外实验诱导成骨程度有显著差异，材料能够调控巨噬细胞分型从而发挥促进成骨的作用，并发现不同生物材料修复的骨组织中组蛋白甲基化酶EZH1表达差异较明显。EZH1通过SET结构域催化H3K27me1和H3K27me2，还能够上调NF-κB下游靶基因表达。本项目拟通过构建巨噬细胞敲除小鼠模型及EZH1敲除鼠，了解EZH1在材料刺激巨噬细胞分型中的作用；并通过免疫共沉淀、ChIP等方法验证巨噬细胞中EZH1调控的下游靶基因及调控机制。本项目通过体内体外实验多角度验证EZH1在骨生物材料进行组织修复过程中的调控机制，使我们更好的规避材料植入后免疫相关的并发症，同时改善生物材料的成骨效果和安全性。

生物材料植入机体后，有利的体内免疫系统应答会在有效的组织再生中起到至关重要的作用，同时避免植入材料的排斥和严重的炎症反应。本项目聚焦在巨噬细胞通过对生物材料的不同反应，向不同亚型极化从而影响材料成骨效果的作用。在研究中我们发现使用骨诱导材料（磷酸三钙，TCP）浸提液处理巨噬细胞后可以显著抑制NF-kappa B（NF-κB）通路的激活，导致EZH1的表达下降。EZH1的减少导致巨噬细胞M1向极化的标记物表达降低，降低了促炎因子的释放；另外，当NF-κB通路被抑制时，EZH1的表达显著下调。进一步的成骨诱导实验表明，这种局部微环境有利于骨再生。随后，继续研究了巨噬细胞极化在不同骨移植材料修复骨缺损中的作用，并在植入物感染控制方面不断尝试，设计了生物纳米材料用于调控巨噬细胞向M2极化并将其应用于骨缺损的修复。将消退素D1（RvD1）或白介素-4（IL4）负载入金纳米笼内或双相磷酸钙支架，可以通过PI3K-AKT和ERK信号通路有效地增强M2巨噬细胞极化，进而可以通过分泌IL-10和TGF-β用于抗炎以及BMP-2直接促进成骨细胞的成骨分化来促进骨生成并防止骨质流失。总之，项目研究了巨噬细胞M2向极化是利于植入物植入后局部组织再生的重要因素，可以通过改变生物材料的表面形貌或控释药物，以调控巨噬细胞M2极化，从而为组织再生提供良好的免疫微环境。