TNF-α/miR-125b调控细胞外基质再平衡对冠状动脉微栓塞后心肌重构的影响及机制

Cardiac remodeling induced by coronary microembolization significantly infulence the clinical prognosis of coronary artery disease after coronary intervention.Synthesis and degradation loss-balance of extracellular matrix plays a critical role in post-injury cardiac remodeling.Presious studies indicatied that microRNA might be involved in regulation of extracellular matrix. However,it is unclear about its effect and detailed mechanism on post-coronary microembolization.Our polit study found that tumor necrosis factor-α(TNF-α) and miR-125b were increased after coronary microembolization. Meanwihle,cardiac fibroblast stimulated by TNF-α,not only upregulated expresssion of miR-125b and synthesis of collagen, but also increased the level of matrix metallopreteinases. Therefore, we supposed that different expressed miR-125b could modulate the re-balance of extracellular matrix after coronary microembolization and improved cardiac remodeling. In this study, mice coronary microembolization model and cardiac fibroblast with differential expression of miR-125b were developed. Then, we try to find out the detailed mechanism between miR-125b and dynamic response of extracellular matrix from the fields of cell differentiation, matrix synthesis and degradation. Meanwhile, luciferase report is applied to document the target genes and signal transduction of miR-125b. Above all, These fingdings will provide novel evidence for clinical treatment of coronary microembolization.

冠状动脉微栓塞所致的心肌重构严重影响冠心病介入治疗的预后。细胞外基质（ECM）合成/降解失平衡是心肌重构的关键。既往研究发现微小RNA（miRs）调控ECM的表达，然而，miRs能否调控微栓塞后多种ECM的再平衡尚不明确。我们前期研究发现，冠脉微栓塞后肿瘤坏死因子α（TNF-α）和miR-125b表达升高；TNF-α体外刺激成纤维细胞，不仅上调miR125b、促进胶原合成，还能促进ECM降解相关蛋白酶（MMPs）的分泌。因此推测，调控miR-125b的表达可以改善微栓塞后心肌重构。本项目拟构建差异性表达miR-125b的冠脉微栓塞小鼠模型和离体成纤维细胞，从成纤维细胞增殖分化、基质合成、基质降解三方面，探索miR-125b不同时相表达对ECM动态合成/降解再平衡的影响。软件预测并以双荧光素酶报告验证miR-125b靶基因，明确信号通路。进而，为临床防治冠状动脉微栓塞后的心肌重构提供新思路。

本次立项课题，按照既定的方向和实验计划，围绕“TNF-α/miR-125b调控细胞外基质再平衡对冠状动脉微栓塞后心肌重构的影响及机制”的主题，从体外细胞实验、在体动物实验和临床队列三方面进行探索和研究。主要的工作汇总及发现成果汇总如下： .（1）首次在国内应用聚乙烯微球成功构建小鼠的非选择性冠状动脉微栓塞模型，并通过病理染色和小动物超声证实模型成果，已发表论文；.（2）完成小鼠冠状动脉微栓塞后的心肌的蛋白组学分析。与假手术组相比，微栓塞组上调蛋白17种，下调蛋白28种；这些差异性蛋白主要的分子功能与NADH脱氢酶活性、氧化还原酶活性等相关；同时，参与的生物过程也主要与细胞能量合成、结构改变等相关，已发表论文。.（3）在冠状动脉微栓塞模型中证实miR-125b在心肌中确实表达上调；同时发现，心肌miR-125b表达与炎症小体NLRP3表达存在关联，离体心肌细胞实验也验证，在TNF-a干预下，伴随着miR-125b的升高的同时，炎症小体有显著性表达；.（4）在心肌缺血状态下，MicroRNA-19a通过下调NHE-1和减少钙超载从而减弱缺氧诱导的心肌细胞凋亡；.（5）完成冠状动脉微栓塞相关的队列研究入组（冠状动脉介入术后的围手术期心肌损伤）1500例，完成血清miR-125b水平检测，发现其升高对预后存在不良影响。.在近4年的课题工作中，发表课题相关性SCI论文9篇；其中，课题负责人均为第一或者共同第一作者参与发表；参加国际会议交流3次；协助培养研究生4人。

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