外泌体源性microRNA对肥胖受损心肌的保护作用机制研究

Exosome-derived miRNA can regulate and control or keep far apart from the target cells' inflammation in autocrine or paracrine processes. Studies have shown that miRNA-210 can affect the gene expression of the transcription factor NF-kB. It has also been found in the author's previous study that obesity myocardial damage is triggered by NLRP3-mediated chronic inflammation and can be reduced by the exosomes. Nothing has been found with the mechanism but there is assumption that the exosome-derived miRNA-210 works well for the reduction through the NF-kB-NLRP3 signal transduction pathway, thereby protecting the damaged obesity myocardium. The present study aims to reveal the mechanism through hyperlipidemia cardiomyocyte culture in vitro. Obese mice injected with myocardial progenitor cell-derived exosomes (CPC-Exo) will be taken as the subject and ultrasound biomicroscopy will be adopted to detect how CPC-Exo-derived miRNA-210 interferes with the subject's myocardial contraction; analysis will then be done for the miRNA-210's effects on the inflammatory factors related to the pathway. It is proposed a new theory and trend of research on the topic.

外泌体源性miRNA可通过自分泌或旁分泌的方式调控自身或远隔靶细胞的炎症反应。据研究miRNA-210可影响NF-kB的基因表达。我们前期研究证实，肥胖心肌损伤主要是通过NLRP3介导的慢性炎症引发，外泌体能下调心肌的炎症反应，但具体机制尚不清楚，推测外泌体源性miRNA-210通过NF-kB-NLRP3信号转导通路下调了炎症反应，从而对肥胖受损心肌进行保护。本项目拟以注入心肌祖细胞源性外泌体(CPC-Exo)的肥胖小鼠为研究对象，应用超声生物显微镜活体检测CPC-Exo源性miRNA-210干扰肥胖小鼠的心肌收缩功能；分析CPC-Exo源性miRNA-210对肥胖小鼠NF-kB-NLRP3信号转导通路相关炎症因子的影响；并通过体外高脂心肌细胞培养，探讨CPC-Exo源性miRNA-210通过调控NF-kB-NLRP3通路保护肥胖受损心肌的机制。旨在为保护肥胖受损心肌提供新的理论依据和靶点

构建肥胖小鼠模型，采用超速离心法提取血浆外泌体，检测外泌体的粒径、形态以及表面标志蛋白，为后续外泌体研究奠定基础。采用高通量测序技术分析肥胖组和正常组小鼠血浆外泌体中的miRNA表达差异，筛选可能和肥胖相关心血管疾病发生密切相关的标志物，初步探索其在疾病发展过程中的生物学作用。验证应变技术对肥胖小鼠左室收缩功能监测的有效性，通过与病理相结合进一步说明肥胖所致心肌损伤改变与炎症因子的关系。 使用高脂饲料构建肥胖模型，数据结果稳定；使用超速离心法可以稳定的提取血浆外泌体，粒径、浓度稳定性佳，拥有典型的双层膜结构，标志蛋白表达稳定,可为后续实验奠定基础。 正常组和肥胖组小鼠血浆外泌体miRNA表达谱之间存在差异,其中显著差异表达的miRNA中，10个为上调，19个为下调，可能通过MAPK和AMPK信号通路，调控肥胖相关的心室重塑、新生血管生成、心肌纤维形成;通过大量查阅文献，miRNA-27a-5p、miRNA-99a-5p对预测早期肥胖相关心肌损伤有潜在的临床价值。高脂饮食诱导的肥胖小鼠心脏发生形态学改变，呈向心性肥厚；UBM可无创检测肥胖小鼠的心脏收缩功能，其应变技术可定量评估肥胖小鼠心肌收缩功能，判断心肌纤维受损程度，NLRP3炎症小体以及 Caspase-1炎症因子参与肥胖小鼠心肌损伤的过程。