树突状细胞的负向免疫调控在心肌损伤与抗损伤中的意义与机制

Inflammation is a complicated balanced process in cardiac injury and protection. Dendritic cells (DC) initiate the immune inflammation response, but the negative regulatory function of DC is critical for the maintenance of immune homeostasis. The negative modulatory function of DC has been reported to alleviate ischemic cardiac injury and the remodeling process. However, how to induce the negative regulatory functions of DC and how they will attenuate the cardiac impairment remain undetermined. microRNAs (miRNAs) are critical for DCs functions, and furthermore, miRNAs are involved in ischemic cardiac injury and anti-injury process. We have found that miR-181a and miR-378 promote the negative regulatory DC and additionally, these two genes are involved in cardiac remodeling process. Therefore, we put forward the hypothesis that miR-181a/378 promote the negative modulatory DC, as as to attenuate the ischemic cardiac injury and to improve the heart functions. To illuminate this point, firstly, we want to demonstrate the negative regulatory functions and the mechanisms of miR-181a/378 to DC as well as their protective roles to the ischemic cardiac injury (named miR DC-neg); then, we try to clarify the protective functions of miR DC-neg to the ischemic heart, based on the following animal models, myocardial infarction mice, the conditionally knock-in mice of miR-181a/378 in DCs, and the DC-depletion mice (flt3-/-) in combination of miR DC-neg-transfution; finally, we want to clarify the clinical significance of miRNA DC-neg in myocardial infarction patients. In summary, through this project, we try to illuminate a novel epigenetic mechanism in ischemic cardiac injury and anti-injury process mediated by miRNA-induced DC negative modulation functions. And we also want to demonstrate the great significance of this mechanism so as to improve the clinical management of the ischemic cardiac injury patients.

炎症在心肌损伤与抗损伤中是复杂的平衡过程。树突状细胞（dendritic cell, DC）始动炎症，而DC负向调控则维持免疫稳态。诱导DC负向调控能减轻心肌缺血损伤与重构，但机制不清。miRNAs是DC的关键调控因素，并参与心肌损伤与抗损伤。我们已发现miR-181a和miR-378诱导DC负向调控并与心肌重构关系密切。因此假说：miR-181a/378诱导DC负向调控以减轻心肌损伤并保护心肌。研究拟先确立miR-181a/378诱导DC负向调控并减轻心肌损伤的作用和机制（miR DC-neg）, 再基于条件性DC过表达miR-181a/378小鼠心梗模型以及DC缺失并回输miR DC-neg，揭示诱导DC负向调控以保护心肌的效应，最后研究人群miR DC-neg与心梗以揭示其临床意义。通过本研究，阐明心肌损伤与抗损伤中miR DC-neg介导的新表观遗传调控机制，并揭示其重要临床意义。

树突状细胞介导的炎症反应在心肌损伤和抗损伤过程中发挥重要作用。在该项目中，我们发现ox-LDL可通过c-Fos通路激活DC并促进炎症反应，而miR-181a则可以减弱ox-LDL对DC的激活及促炎作用。在上述研究的基础上，我们采用心肌坏死液刺激DC，发现DC会被激活，而激活状态的DC则对心肌细胞有明显的促凋亡作用。进而，我们发现在DC中过表达miR181a/150会显著抑制DC对心肌细胞的凋亡作用。另外，我们针对DC进行了一些其他方面的探索性研究。发现，胰岛素可刺激DC成熟并增强对ox-LDL的摄取，从而激活DC并促进炎症反应；我们还发现，Ang 2可促进DC的成熟及诱导炎症反应从而促进AS，而使用阿托伐他汀可通过PI3K/Akt/Nrf 2通路抑制该效应从而发挥抗AS作用。在针对miRNA进行研究时，我们还发现外泌体（exosomes）是miRNA在细胞间转移和发挥作用的重要媒介，我们发现小鼠MI后心梗部位浸润的DC显著增加，并能通过分泌外泌体而激活CD4 T细胞，同时体内研究表明，体外注射外泌体可以显著改善小鼠MI后心脏功能。DC分泌的外泌体可被CMEC摄取，使CMEC的VEGF表达增高并增强CMEC的成管作用，同时发现外泌体内miR-126、miR-23a、miR-150、miR-16表达增高，而既往研究表明这些miRNA可促进血管新生。成熟DC分泌的外泌体可以导致人脐静脉内皮细胞（HUVEC）高表达VCAM、ICAM及E-selectin等黏附因子，促及粥样硬化进展，该现象通过外泌体膜表面的TNF-α与HUVEC的受体直接结合诱导NF-κB途径激活完成。MI后小鼠血脂水平增高，肝脏DC浸润增加,肝脏浸润的DC可通过MTOR-AMPK-SREBP通路影响脂质代谢，使血脂水平增高而促进AS发生。成熟DC外泌体可以诱导非成熟DC 变为成熟，也可以导致T细胞高表达炎症因子。体内注射外泌体后，成熟外泌体更多的富集于脾脏，并且可以引起小鼠高表达DC及T细胞相关的炎症因子。成熟外泌体富集于脾脏通过DC外泌体表面的CCR-7及其受体完成。我们的研究结果提示，树突状细胞在心梗后心肌重塑、动脉粥样硬化乃至血脂代谢过程中发挥了复杂的作用，其作用机制包括miRNA、外泌体等。针对上述作用机制的更深入研究有助于我们发现治疗疾病的新靶点。