丹红注射液通过外泌体miR-126调控心脏祖细胞Ras/ERK通路干预心梗后心肌修复的机制研究

Cardiac progenitor cells (CPC) play an important role in myocardial repair after myocardial infarction. How to improve CPC function is the hot focus of cardiovascular regenerative medicine research. After the ischemic event, endothelial cells secrete a large number of exosomes containing miR-126. These exosomes activate the Ras / ERK pathway in CPC through the paracrine mechanism. Danhong injection, a typical TCM preparation for promoting blood circulation, can effectively improve the long-term prognosis of patients with myocardial infarction after PCI and heart failure. Our previous study found that， after myocardial infarction, Danhong injection had the effects of restoring damaged heart function, reducing infarct size, increasing expression of CPC marker protein Sca1, correlated with the selectively increased level of miR-126. Therefore, we put forward the hypothesis: “Danhong injection can regulate the expression of exosomal miR-126, activate the Ras/ERK pathway in CPC, improve CPC function and myocardial repair.” To study the cellular and molecular mechanism of Danhong injection in promoting CPC function and myocardial repair, myocardial infarction mouse, exosomes and CPCs are designed as subjects of this research. To explore the value of Danhong injection in cardiovascular regenerative medicine, the effect of Danhong injection combined with CPC transplantation on myocardial infarction mouse will be also investigated.

心脏祖细胞（CPC）在心梗后心肌修复中起着重要作用。如何提高CPC功能是目前心血管再生医学研究的重点、难点问题。缺血事件发生后内皮细胞分泌大量含有miR-126的外泌体，通过旁分泌机制激活CPC Ras/ERK通路调控其功能。丹红注射液是活血化瘀代表中药制剂，可有效改善心梗PCI术后患者及心力衰竭患者的远期预后。我们前期研究发现丹红注射液可恢复心梗后受损心功能，缩小梗死面积，增加CPC标记蛋白Sca1表达，选择性提高miR-126表达水平。据此提出丹红注射液通过调节心梗后外泌体miR-126表达水平，激活CPC Ras/ERK通路，提高CPC功能，进而促进心肌修复的假说；本课题以心梗模型小鼠、外泌体、CPC为研究对象，研究丹红注射液调节CPC功能促进心肌修复的细胞及分子机制，并通过丹红注射液联合CPC移植干预心梗模型小鼠的实验方案，探讨丹红注射液在心血管再生医学中的应用价值。

外泌体miRNA是近年抗心肌损伤药物治疗的研究热点。心梗后内皮细胞可分泌大量含有miRNA的外泌体，通过旁分泌机制促进细胞增殖、分化并释放促血管新生因子。丹红注射液是活血化瘀中药的代表制剂，临床应用治疗心梗PCI术后和心梗后心力衰竭患者安全、有效。本研究成功建立了小鼠心梗模型、主动脉缩窄致心肌肥厚模型，原代小鼠心脏微血管内皮细胞和小鼠间充质干细胞分离、纯化及培养，证实了“丹红注射液通过调节心梗后外泌体miR-126表达水平，激活心脏祖细胞Ras/ERK通路，提高心脏祖细胞和内皮细胞功能，进而促进心肌修复”的假说。我们的研究发现：（1）丹红注射液可上调心肌梗死后心脏miR-126/Spred-1/ERK/VEGF通路促进心脏祖细胞增殖和存活率，促进心梗后血管新生，改善心肌功能；（2）丹红注射液可通过上调缺氧内皮细胞miR-126/Spred-1/ERK/VEGF通路促进内皮细胞增殖、迁移；（3）丹红注射液诱导的内皮细胞外泌体中miR-125b、miR-126表达有所升高，是调控心脏干（祖）细胞功能的主要miRNA；（4）丹红注射液诱导的外泌体可通过miR-126/Spred-1/ERK通路促进间充质干细胞增殖、迁移；（5）丹红注射液外泌体miR-125b可能通过调控Rassf1基因甲基化延缓TAC术后小鼠心肌纤维化。经过三年的研究工作，基本完成了预定研究目标：（1）阐明了丹红注射液通过内皮外泌体miR-126调控的心脏祖细胞功能，激活miR-126/ERK/VEGF通路，实现心肌修复和血管新生的细胞及分子机制，为应用丹红注射液干预心梗预后提供实验依据；（2）从外泌体miRNA角度入手，发现miR-126、miR-125b是丹红注射液诱导外泌体中主要的功能性miRNA，为活血化瘀中药促血管新生机制研究提供新思路、新方法；（3）研究丹红注射液或丹红注射液诱导的内皮细胞外泌体对干（祖）细胞功能的调控作用和机制，探讨丹红注射液在心血管再生医学中的应用价值。（4）从miR-125b调控的DNA甲基化角度入手，研究了丹红注射液干预心肌肥厚和心室重构的分子机制，为丹红注射液干预心肌肥厚和心室重构提供了实验依据。