基于泡沫细胞源性外泌体microRNA介导的细胞间通讯探讨冠心病痰瘀互结证的发病机制及痰瘀同治法的干预作用

The location of chest pain was in heart, the return of heavy and turbid part of food essence to heart causing collaterals injury and blood stagnating, eventually leading to stasis of phlegm and blood and heart vessel blockage. MiRNA derived from exosomes has specificity of tissues and could be used as a messenger mediated communication between cells by targeting tissues and organs to regulate their function. Because of its specificity and temporal expression is extremely similar with dynamic and temporal characteristics of TCM syndrome, miRNA derived from exosomes could reflect the essence of the TCM syndrome. This study was intended to take the patients with phlegm and blood stasis syndrome of coronary heart disease as the research objects , starting from epigenetics and macrophage differentiation pathways and using gene sequencing, flow cytometry, Western blot to identify the pathogenesis of foam cell-derived exosomal microRNA regulating the expression of endothelial inflammatory factor, promoting the smooth muscle cell proliferation and thus accelerating the formation of atherosclerosis plague, and reveal the nature of phlegm and blood stasis syndrome of coronary heart disease. At the same time, using rat models with phlegm and blood stasis syndrome of CHD and in vitro cell for experiment to further clarify the molecular mechanisms of foam cell-derived exosomal microRNA regulating the vascular microenvironment in patients with phlegm and blood stasis syndrome of CHD and explore the molecular mechanism of the method of removing phlegm and blood stasis in the CHD treatment from the angle of foam cell-derived exosomal microRNA regulating the vascular microenvironment, and thus reveal the main targets for the method of removing phlegm and blood stasis in the regulation of phlegm and blood stasis syndrome of CHD, It will no doubt be of great significance for guiding the clinical treatment of CHD, and lay a foundation for finding a more effective prescription for CHD.

胸痹心痛病位在心，过食膏梁厚味，浊气奎遏，蕴湿生痰，伴之络脉损伤、瘀血内停，最终导致痰瘀互结、心脉痹阻。外泌体源性miRNA具有组织细胞特异性，可作为信使介导细胞间通讯，靶向调控组织器官功能，因其特异性和时序性与中医证候的动态时空特征极其相似，可反映中医病证的实质。本项目拟以临床冠心病痰瘀互结证患者为研究对象，从表观遗传学及巨噬细胞分化途径入手，运用基因测序、流式、免疫印迹等技术，明确痰瘀互结证患者泡沫细胞源性外泌体miRNA调控内皮细胞炎症因子表达、促进平滑肌细胞增殖，加速动脉粥样硬化斑块形成的发病机制，从而揭示冠心病痰瘀互结证的证候本质。同时应用病证结合大鼠模型及细胞实验，进一步阐明泡沫细胞源性外泌体miRNA对血管微环境的分子网络调控机制，揭示“痰瘀同治法”调控冠心病痰瘀互结证的主要作用靶点，从血管微环境调控角度探讨“痰瘀同治法”的分子调控机制，从而为寻找更加有效的治疗方药奠定基础。

胸痹心痛病位在心，过食膏梁厚味，浊气奎遏，蕴湿生痰，伴之络脉损伤、瘀血内停，最终导致痰瘀互结、心脉痹阻。外泌体源性miRNA具有组织细胞特异性，可作为信使介导细胞间通讯，靶向调控组织器官功能，因其特异性和时序性与中医证候的动态时空特征极其相似，可反映中医病证的实质。本项目分别以冠心病大鼠、LDLR-/-基因敲除小鼠、ApoE-/-基因敲除小鼠冠心病（动脉粥样硬化）模型及人晚期动脉粥样硬化斑块中巨噬细胞、ox-LDL诱导人THP-1细胞构建泡沫细胞模型及痰瘀互结证冠心病患者血清外泌体miRNA为研究对象，从表观遗传学及巨噬细胞分化途径入手，运用基因测序、流式、免疫印迹等技术，明确痰瘀互结证冠心病患者血清外泌体特异性miRNA、巨噬细胞分子共表达调控网格，构建ceRNA分子共表达网络。通过冠心病（动脉粥样硬化）巨噬细胞的转录和异质性确定痰瘀同治法治疗冠心病痰瘀互结证的有效作用靶点，说明冠心病痰瘀互结证发病机制，从而揭示冠心病痰瘀互结证的证候本质。我们的研究结果表明，痰瘀互结证冠心病患者血清外泌体miRNA表达具有特异性，差异基因主要集中在炎症、氧化应激、免疫反应、脂类代谢等通路上。同时我们对冠心病患者血清外泌体miRNAs测序结果表明有14种miRNAs与健康人群存在显著差异，通过调控Ctss、Ccr2及Trem2等炎性因子表达引起动脉微环境的改变。在动脉粥样硬化斑块中发现5个巨噬细胞群，在脂质代谢和分解代谢中发挥了不同的功能，揭示了动脉粥样硬化斑块中巨噬细胞的转录特性和异质性。应用基因芯片对冠心病痰瘀互结证患者血清RNA测序分析，构建ceRNA分子共表达网络，揭示冠心病痰瘀互结证发病机制，探讨痰瘀同治法的有效作用靶点，从ceRNA网络调控冠脉微环境角度基本揭示冠心病痰瘀互结证的证候本质。我们还通过活血中药丹参、三七，化瘀袪痰经方二陈汤、瓜蒌薤白半夏汤明确了痰瘀同治法治疗冠心病痰瘀互结证的有效作用靶点，从外泌体miRNA调控冠脉微环境角度基本揭示冠心病痰瘀互结证的证候本质。从而为寻找更加有效的治疗方药奠定基础。