心肌细胞外泌体lncRNA-TUG1调控缺血再灌注损伤后心脏纤维化和重构机制研究

Most of the patients in the treatment of ischemic heart disease have myocardial ischemia-reperfusion injury, which will induce cardiac fibroblast proliferation and cardiac remodeling. So how to improve the heart's anti-ischemia reperfusion injury and reduce the occurrence of cardiac remodeling become one of the focuses of treatment of ischemic heart disease. Exosomal long non-coding RNAs (lncRNAs) mediate cellular communication between cardiomyocytes and fibroblasts via paracrine manner. Our previous studies found that exosomes secreted from ischemia-reperfusion injured myocytes by activation fibroblasts and significantly downregulation SPRY1. Meanwhile, the results of RNA-Seq and bioinformatical analysis indicated that exosomal lncRNA-TUG1 released from from ischemia-reperfusion injured myocytes could promote cadicac fibrosis via regulating SPRY1. With the knockdown of TUG1, the exosomes released from ischemia-reperfusion injured myocytes could not activate myofibroblasts and downregulate the expression of SPRY1. Therefore, we hypothesize that TUG1 derived from myocytes could downregulate SPRY1 expression and activate cardiac fibroblasts by connecting the miR-21 or the interaction protein, which eventually contribute to cardiac fibrosis or remodeling. The present study mainly focuses on the following three aspects: ①to verify the TUG1 released from cardiac myocytes could enter into and activate cardiac fibroblasts; ②to reveal the underlying modulatory mechanisms of exosomal TUG1 on SPRY1 expression; ③to elucidate the role of exosomal TUG1 secreted by cardiac myocytes in cardiac fibrosis and occurrence of fibrillation in vivo. Our work may provide new insights into the potential role and molecular mechanisms of exosomes in regulating cardiac fibrosis and remodeling induced by ischemia-reperfusion injury. Our result will make an important method to reduce cardiac fibrosis and remodeling induced by ischemia-reperfusion injury.

大部分缺血性心脏病患者在治疗时都存在缺血再灌注损伤诱发的纤维细胞增生和心脏重构，如何减少纤维化和重构的发生成为缺血性心脏病治疗的难点之一。外泌体可负载lncRNA以旁分泌途径实现心肌细胞与成纤维细胞间信息交流。前期研究发现：缺血再灌注损伤后心肌细胞释放的外泌体可激活成纤维细胞，使其抑制因子SPRY1表达明显减少；测序和生物信息学提示：外泌体lncRNA-TUG1通过调控成纤维细胞SPRY1表达，引起心脏纤维化。我们提出假设：缺血再灌注损伤时心肌细胞源性外泌体TUG1通过结合成纤维细胞内miR-21或互作蛋白调控SPRY1表达，促进心脏纤维化。本项目拟从细胞、动物和临床标本三方面阐明外泌体TUG1调控心脏成纤维细胞SPRY1引起心脏纤维化，促使心脏重构发生的分子机制。研究结果将为优化缺血再灌注心脏抗损伤能力，减轻因缺血再灌注损伤后引起的心脏纤维化和重构开阔新视野。

伴随着全球人口老龄化问题的凸显，老年性疾病已成为主要的发病种类和死亡原因，其中缺血性心脏病尤为显著。缺血性心脏病可引发心肌的不可逆性坏死，功能性心肌细胞减少，纤维细胞增生，瘢痕形成以及心脏结构改变，最终导致难以控制的心力衰竭和死亡。本研究通过结扎冠状冠状动脉左前降支成功建立了缺血再灌注细胞模型，并验证心肌细胞外泌体TUG1可进入并激活心脏成纤维细胞。通过慢病毒载体构建TUG1过表达原代心脏成纤维细胞，给予慢病毒沉默SPRY1或过表达miR-21，检测了miR-21和SPRY1在TUG1激活成纤维细胞中的作用及机制。并在成纤维细胞中验证TUG1、miR-21和SPRY1的结合关系。同时与德克萨斯大学健康科学中心和贝勒医学院进行了学术交流。收集缺血再灌注损伤患者临床资料、血液样品，超声记录心脏功能和形态改变，并对患者进行了随访，提取外泌体并验证了患者TUG1及心脏组织纤维化指标的相关性。在大鼠缺血再灌注损伤大鼠模型中，通过心脏局部注射慢病毒沉默TUG1，验证了沉默TUG1对纤维化的逆转作用，为未来心脏缺血再灌注损伤提供新的治疗方法提供了重要依据。