丝胶蛋白通过细胞外囊泡调控Hippo信号通路促进心肌再生的机制研究

Myocardial infarction (MI) is one of the most important diseases that cause human death. Considering that the adult human cardiomyocyte has a very limited capacity to proliferate, current therapies for MI are ineffective in repairing infarcted myocardium. Therefore, exploring the approach which facilitate regeneration of injured cardiac tissue is highly desired. Recently, we reported the injection of in situ forming crosslinked sericin hydrogel promotes heart functional recovery accompanied with reduced inflammatory responses, attenuated apoptosis and promoted angiogenesis in mouse infarcted heart. In this study, we found direct intramyocardial injection of sericin increased the number of Ki67-positive, pH3-positive, and BrdU-positive cardiomyocytes at the infarcted border zone, promoted YAP accumulated in the nucleus, and ameliorated cardiac function in the adult mouse heart. In vitro results indicated that sericin stimulated the production of extracellular vesicles (EVs) in nonmyocytes. Then the EVs induced YAP to translocate into nucleus and increased the number of cardiomyocytes. Thus, we raise the hypothesis that sericin induces EVs secretion in nonmyocytes, and the EVs-packaged key biological molecules regulate Hippo signaling pathway, leading to cardiac regeneration after MI. We will employ MALDI-TOF mass spectrometry and miRNA-seq to find out EVs-carried key biological molecules involved in cardiomyocytes proliferation and then explore the mechanisms involving the nucleus translocation of YAP by using integrated approaches including molecular cell biology and animal model. This project focuses on clarifying the mechanisms that sericin promotes cardiac regeneration, which may provide a theoretical basis and potential targets for the clinical treatment of MI.

心肌梗死是造成人类死亡的重要疾病之一，因成年人心肌细胞极难增殖，目前治疗手段无法修复已梗死心肌，因此亟待寻找心肌再生的方法。我们前期发现丝胶在交联剂帮助下能在心肌注射区原位成胶，通过促进血管生成及抑制心梗区炎症反应和凋亡来改善小鼠心功能。本研究中我们发现向心肌直接注射丝胶蛋白可增加成年小鼠心梗周边区Ki67、pH3及BrdU阳性心肌细胞数，促进YAP进入细胞核，改善心功能；体外实验证实丝胶蛋白诱导非心肌细胞分泌细胞外囊泡，该囊泡促进YAP入核及心肌细胞增殖。据此我们提出假设：丝胶蛋白通过诱导非心肌细胞产生细胞外囊泡，调控Hippo信号通路，促进心梗后的心肌再生。我们将使用质谱及miRNA-seq筛选囊泡携带的诱导心肌细胞增殖的关键生物分子，并利用分子细胞生物学及动物模型等技术方法探索其影响YAP入核的机制。本研究将阐明丝胶蛋白促进心肌再生的分子机制，为心梗的临床治疗提供理论依据和潜在靶标。

心脏病是全球首要死因，冠状动脉疾病可导致冠状动脉急性闭塞，造成心肌梗塞和数百万心肌细胞的死亡。不幸的是，成年人的心脏几乎没有再生能力，梗塞的区域被基本没有心肌细胞的纤维化疤痕所取代，这导致病情逐步进展为心力衰竭，预后变差。本项目探索丝胶这一天然生物材料促进心肌细胞损伤修复的分子机制，发现丝胶蛋白能够诱导非心肌细胞分泌细胞外囊泡，该囊泡进入心肌细胞后抑制胞浆内miR195表达，促进Yap蛋白富集从而诱导心肌细胞增殖和心功能恢复。基于此我们设计并制备了携带miR195核酸海绵的工程化细胞外囊泡，它能够靶向损伤的心肌层并以膜融合的方式进入心肌细胞，通过清除胞浆内的miR195诱导心肌细胞增殖修复。本研究发现了心肌再生相关的新靶标分子，并开发了针对性的核酸药物递送载体，有广阔的应用前景。此外，本项目也探索了丝胶生物材料在肌肉修复中的应用及分子机制，新发现Cdc20-Mef2c信号通路参与肌肉的再生，为心肌、骨骼肌的创伤修复提供理论依据。