弱酸性富钙磷离子环境下单核细胞来源外泌体的miRNA表达变化及其靶细胞效应研究

The process of osseointegration and bone remodeling is usually initiated by the recognition of monocyte, and followed by the activities of osteoclasts and osteoblasts. The intercellular dialogue mostly mediated by varies cytokines and inflammatory signals. Exosomes was found to be capable of carrying protein, mRNA, and miRNA, is considered to be a new cell communication medium, the role of exosome in the procedure of osseointegration and bone remodeling remain unclear so far. .Our previous studies indicated that calcium phosphate cement (CPC)/polylactide-glycolic acid (PLGA) microspheres composite has a favorable bone ingrowth and bone replacement effect, however, bone ingrowth is limited to the area where degradation of PLGA microspheres are completed. We clarified that lactic acid, the PLGA degradation production, have a synergic effect with the local rich calcium and phosphate ions environment, in the activation of osteoclast and coupling of osteoblast via RANK-RANKL-OPG pathway. Our hypothesis for the project is the monocyte derived exosomes participate in the cross talk between bone remodeling related cells by alteration its miRNA profile. The project will collect exosomes secreted by mononuclear cells when treated with a variety of acidic rich calcium- and phosphorus- ions condition，expression profile of exosomal miRNAs will be analyzed by FACS and high-throughput sequencing technology. bone marrow mesenchymal stem cells (BMSCs) and precursor monocyte will be treated with exosomes collected in the condition mentioned before respectively, its effect on differentiation of target cells will be observed by using Laser scanning confocal microscope、RT-PCR、western blot.

生物材料与骨的整合多由单核细胞识别启动，偶联破骨细胞和成骨细胞活动完成。这些细胞活动的对话机制除了涉及多种细胞因子和炎性信号通道之外，外泌体的作用还有待明确。前期工作已发现磷酸钙骨水泥/聚乳酸-羟基乙酸共聚物(PLGA)微球复合物有明显的降解成骨效应，但成骨局限于PLGA降解部位。已证明该复合物可产生弱酸性，富钙、磷离子环境，通过RANK-RANKL信号加强破骨细胞活动，并偶联成骨活动。本项目假说是单核细胞源性外泌体通过其miRNA表达变化参与了上述过程。研究拟收集单核细胞在模拟材料降解环境下向多核巨噬细胞样细胞分化过程中产生的外泌体，利用高通量测序技术分析其外泌体miRNA的表达谱，预测靶基因及其调节网络。将各组外泌体分别孵育刺激骨髓间充质干细胞（BMSCs）和破骨前体细胞，利用激光共聚焦显微镜、RT-PCR、western blot等手段观察其对靶细胞的分化效应，以验证本假说。

项目背景：生物材料与骨的整合多由单核细胞识别启动，偶联破骨细胞和成骨细胞活动完成。这些细胞活动的对话机制除了涉及多种细胞因子和炎性信号通道之外，外泌体的作用还有待明确。前期工作已发现磷酸钙骨水泥/聚乳酸-羟基乙酸共聚物(PLGA)微球复合物有明显的降解成骨效应，但成骨局限于PLGA降解部位。已证明该复合物可产生弱酸性，富钙、磷离子环境，通过RANK-RANKL信号加强破骨细胞活动，并偶联成骨活动。本项目假说是单核细胞源性外泌体通过其mi RNA表达变化参与了上述过程。.主要研究内容：研究拟收集单核细胞在模拟材料降解环境下向多核巨噬细胞样细胞分化过程中产生的外泌体，利用高通量测序技术分析其外泌体miRNA的表达谱，预测靶基因及其调节网络，利用质谱仪检测外泌体蛋白质，并进行蛋白质组学分析。弱酸性环境处理刺激上皮细胞和破骨前体细胞， 利用激光共聚焦显微镜、RT-PCR、western blot等手段观察其对靶细胞的分化效应，以验证本假说。.重要结果和关键数据：.（1）10mmol/L乳酸为促小鼠RAW264.7细胞破骨向分化的最适浓度。在10mmol/L乳酸微环境下，单核细胞产生的外泌体miRNA中mmu-let-7b-5p，mmu-miR-712-5p和 mmu-miR-3084-3p的靶基因与破骨细胞分化相关。 .（2）CPC/PLGA复合降解液可以增强小鼠RAW264.7细胞破骨相关特异性蛋白的表达。miR-3473b、mmu-miR-3090-5p、mmu-miR-346-3p和mmu-miR-714miRNA可能是复合降解液影响单核巨噬细胞向破骨细胞分化的重要调节因子。.（3）miR-210-3p–FGFRL1-PI3K/ATK信号轴是乳酸调节血管新生的重要途径之一，乳酸通过信号轴调控VEGF和FGF1的表达，促进血管内皮细胞的迁移能力和成管能力。.（4）推测乳酸作用下破骨细胞来源外泌体与内皮细胞增殖有调节作用的相关蛋白有8个，分别是Tnf、ALDH2、Atp5f1a、Comp、Thbs4、Rptor、Aldh1a7、Kng1。差异蛋白参与的信号通路包括：MAPK信号通路、NF-Kappa B信号通路、mTOR信号通路、PI3K-Akt信号通路等。.科学意义：为进一步阐明CPC/PLGA降解成骨过程中细胞间的交互对话机制提供科学依据。