肥大心肌细胞来源胞外体在TLR7介导的心肌炎症反应调节中的作用及机制

Cardiac hypertrophy was found to be associated with several cardiovascular diseases, which seriously threaten people’s health. The exosome-mediated inflammatory pathway was proposed to play an important role in the occurrence and development of cardiac hypertrophy, but the molecular mechanisms remain poorly understood. Our results showed that the expression of miR-29a was significantly enhanced in exosomes derived from hypertrophic cardiac myocytes induced with angiotensin II. However, the miRNA expression profile in such exosomes is currently unknown. In a preliminary experiment we further found that exosomes derived from hypertrophic cardiac myocytes were capable of activating Toll-like receptors 7 (TLR7) in macrophages. Recently, emerging data have indicated that TLR7 was an important regulator of cardiac inflammation. Therefore, we suppose that cardiac hypertrophy leads to changes of exosomes derived from hypertrophic cardiac myocytes in miRNA content, which contribute to inflammatory response in a TLR7 mediated manner. The aim of this study is to detect the relevant properties of exosomes include size, morphology, production and miRNA content using transmission electron microscope and miRNA microarray. Also, the effect of exosomes on cardiac inflammation and its possible mechanism will be investigated at whole body, organ and cellular levels using laser confocal microscope, flow cytometry and co-Immunoprecipitation. This work has the potential to bring new ideas and targets for the successful prevention and treatment of cardiac hypertrophy and heart failure.

心肌肥大与多种心血管疾病相关，严重危害人类健康。胞外体介导的炎症通路在心肌肥大发生发展中作用关键，但机制不详。我们研究显示，血管紧张素II诱导的肥大心肌细胞来源胞外体中miR-29a表达明显增加，但其具体的microRNAs（miRNAs）表达特征尚不清楚。在预实验中，我们发现：肥大心肌细胞来源胞外体能活化巨噬细胞TLR7信号通路。资料表明，TLR7是心肌炎症反应的重要调控因子。因此，我们推测：肥大心肌细胞来源胞外体携带的miRNAs表达发生变化，并经TLR7影响心肌炎症反应。本项目拟应用透射电子显微镜、基因芯片等技术研究肥大心肌细胞来源胞外体大小、形态、分泌量、miRNAs表达等特征，进一步采用激光共聚焦显微镜、流式细胞仪、免疫共沉淀等技术，从整体-器官-细胞层次观察肥大心肌细胞来源胞外体对心肌炎症反应的影响，并探讨其分子机制。此研究将为心肌肥大和心力衰竭的防治提供新思路和新靶点。

心肌肥大与多种心血管疾病相关，严重危害人类健康。外泌体介导的炎症通路在心肌肥大发生发展中作用关键，但机制不详。本项目首先建立心肌肥大细胞模型，分析肥大心肌细胞来源外泌体中miRNAs的表达变化情况，同时探讨其大小、形态、分泌量等特征。与对照组相比，模型组外泌体数量明显增加[(8.80 ± 1.48)个比(3.80 ± 0.84)个，t = 6.57，P < 0.05]，平均直径明显增加[(75.05 ± 3.24) nm比(60.11 ± 3.86) nm, t = 8.39，P < 0.05]，表面积明显增加[(4547.05 ± 398.46) nm2比(2910.76 ± 369.83) nm2, t = 8.51，P < 0.05]，圆形度明显降低[(0.89 ± 0.02)比(0.98 ± 0.01)，t = -9.62，P < 0.05]，提示肥大心肌细胞来源外泌体与正常心肌细胞分泌的外泌体相比在数量及形态上发生了明显的改变。与对照组相比，肥大心肌细胞来源外泌体差异表达miRNAs共14个，其中13个miRNAs表达上调，仅1个miRNA表达下调。差异表达miRNAs靶基因参与多个与心肌肥大相关的信号通路，包括：MAPK信号通路、ErbB信号通路、PI3K-Akt信号通路、Wnt信号通路、Hippo信号通路等，进而通过这些信号通路调节心肌细胞增殖分化、炎症反应等病理生理过程。采用肥大心肌细胞来源外泌体对进行干预，从动物水平证实了肥大心肌细胞来源外泌体对心肌炎症反应具有调节作用。进一步研究发现，差异表达miRNAs通过调节TLRs信号通路影响心肌炎症反应，阐述了肥大心肌细胞来源外泌体经TLRs影响心肌炎症反应的分子机制。另外，由于凋亡是机体调控炎症反应的发展和防止炎症后组织管官损伤与瘢痕形成的重要机制，本研究也探讨了miR-139-3p与心肌细胞凋亡的关系及可能机制；而且，鉴于Notch 信号通路参与调节炎症和氧化应激中的关键作用，本研究还发现，miR-151-5p可经Notch等信号通路调控心肌细胞炎症反应，这些结果将为肥大心肌细胞来源外泌体调节炎症通路的研究提供有益的思路启示。综上所述，此研究将为心肌肥大和心力衰竭的防治提供新思路和新靶点。