miR-223调控血小板活化在动脉粥样硬化中的作用及机制研究

Platelets are involved in vascular homeostasis and inflammation through interaction with circulating blood cells and vascular wall. MicroRNAs (miRNAs) are small, conserved and non-coding RNA molecules, which interact directly with specific mRNAs regions regulating gene expression. In recent years, the specific expressions of miRNAs related to platelet activation have become a new viewpoint of atherosclerosis research. Our previous studies have found that miR-223, expressed highly in platelets of the patients with coronary heart disease (CHD), was remarkably correlated with the platelet activation. The results have indicated that the platelet activation regulated by miR-223 may play a novel role in the development of atherosclerosis. Therefore, we will study the role of in platelet activation and atherosclerosis from molecular, cellular, organic, clinical aspects through platelets, vascular endothelial cells and atherosclerosis animal model. Furthermore, we will study the interaction of miR-223 and target genes in atherosclerosis and its mechanism. Firstly, we will study the biological effects of miR-223 on platelet activation, interaction of platelet to endothelial cells and its mechanism from cellular level. Secondly, we will observe whether platelet treated with miR-223 would slow the pathological process of atherosclerosis in atherosclerosis model mouse from organic level. Finally, we will observe the expression change of miR-223 in platelet of patients with coronary heart disease from clinical level. In summary, this study will offer a new therapeutic target and direction for prevention and cure of atherosclerosis; will lay a foundation of revealing the mechanism of platelet activation in atherosclerosis from a new perspective of miR-223 expression related to platelet activation; will provide a new idea for prevention, early diagnosis and treatment of atherosclerosis related diseases.

近年来，与血小板活化相关的微小RNA表达成为动脉粥样硬化（AS）研究的新视点。我们前期研究发现冠心病患者血小板miR-223表达增高，其水平与血小板活化相关，结果提示miR-223调控血小板活化在AS发生发展中可能发挥重要作用。为此，我们将通过血小板、血管内皮细胞和AS动物模型，从分子、细胞、整体、临床四个水平研究miR-223调控血小板活化在AS中的作用，探讨miR-223与靶基因的相互作用在AS发生中的作用机制。细胞水平观察miR-223对血小板活化，对血小板与血管内皮细胞黏附的生物学作用及其机制；整体水平观察直接AS小鼠体内注射经miR-223处理的血小板能否延缓AS病理进程；临床水平观察miR-223与冠心病患者血小板活化因子的关系。本研究将从与血小板活化相关的miR-223特异性表达这个新视角，为揭示血小板活化在AS发生中的作用机理奠定基础，为AS防治提供新的治疗靶点和研究方向。

近年来，与血小板活化相关的微小RNA特异性表达成为动脉粥样硬化研究的新视点。研究发现血小板细胞中miR-223表达最高，并且参与了血小板活化，但是对miR-223调控血小板活化在AS中的作用及其机制还知之甚少。为此，本项目通过血小板、血管内皮细胞和AS动物模型，从分子、细胞、整体、临床四个水平，研究miR-223在血小板活化和动脉粥样硬化中的作用，并且探讨miR-223与靶基因的相互作用。研究发现与非冠心病患者相比，冠心病患者血小板活化标志物CD31和PAC-1阳性表达率显著升高，血小板miR-223表达水平明显升高，而P2Y12 mRNA的表达水平明显降低；血小板miR-223的表达水平与P2Y12的表达水平呈负相关，与CD31、PAC-1的表达水平呈正相关；冠心病患者血小板miR-223的表达水平与Gensini评分呈显著正相关。体外不同浓度ADP刺激血小板后，血小板表面活化标志物CD31、PAC-1阳性表达率显著增加，同时血小板释放的血小板微囊泡表达量显著增加，且呈浓度依赖性；同时发现活化程度越强，血小板中miR-223表达水平降低越显著，而相应血浆中的miR-223表达水平显著升高。双荧光素酶报告基因实验证实miR-223靶向作用于P2Y12 mRNA 3’UTR区。以miR-223的模拟物和抑制物转染血小板，发现miR-223 与P2Y12 mRNA 表达水平呈显著负相关，结果进一步验证miR-223与P2Y12 的相互作用。体内动物实验成功建立动脉粥样硬化小鼠模型。由于转染后血小板活性明显降低，这对后续的血管内皮细胞黏附实验的结果产生不利影响，不能保证动物实验中需要的转染血小板的质量和数量。以上研究结果提示血小板miR-223与血小板活化和P2Y12关系密切，血小板miR-223水平可能成为评估冠状动脉严重程度的指标。不同浓度ADP可以诱导血小板不同程度的活化，血小板释放的miR-223与血小板活化相关，其水平可能成为血小板活化状态的一个监测指标。P2Y12是miR-223的靶基因，miR-223可能通过P2Y12发挥其调控血小板功能的作用。本研究从与血小板活化相关的miR-223表达这个新视角为揭示血小板活化在动脉粥样硬化发生中的作用机制提供了实验依据，为动脉粥样硬化相关疾病的预防、早期诊断及治疗提供新的思路。