外泌体介导下MicroRNA-21调控血管钙化的机制和功能研究

Vascular calcification is an important pathological manifestation of atherosclerosis in the elderly and in diabetic patients. Vascular calcification increases the stiffness of blood vessels, decreases vascular compliance, and significantly increases the risk of cardiovascular accidents. Alteration of gene expression and abnormal differentiation of endothelial progenitor cells (EPCs) are the major causes of intimal calcification. Our preliminary study showed that, in diabetic patients, the calcification related proteins were up-regulated in EPCs and the expression of miR-21 in plasma exosomes was increased. In addition, the diabetic milieu can induce the release of exosomes, which results in the reduction of miR-21 and increase of its target gene WNT1 to regulate EPCs differentiation. We also found that the inhibition of miR-21 expression in EPCs can significantly trigger differentiation of EPCs into osteoblast-like cells. Thus, we hypothesize that exosomes can promote the transportation of miR-21 to extracellular space in diabetes, which up-regulates the intracellular expression of its target genes to cause vascular calcification. In this study, we plan to establish the roles and mechanisms of miR-21 in regulation of vascular calcification and clarify the functions and mechanisms of miR-21 transportation by exosomes. We will also use in vivo experiments to confirm the role of miR-21 in vascular calcification. Finally, clinical research will be performed to see whether miR-21 can be used as a biomarker to predict cardiovascular events. This study will provide a novel therapeutic target and theoretical basis for the prevention of vascular calcification and treatment of cardiovascular diseases.

血管钙化是老年人和糖尿病患者动脉粥样硬化的重要病理表现；钙化增加血管硬度，降低血管顺应性，显著增加心血管危险。内皮祖细胞(EPCs)基因表达改变和分化异常是血管内膜钙化的主要原因。我们前期研究发现糖尿病患者EPCs钙化相关蛋白表达增加，血浆外泌体miR-21表达增加；糖尿病环境促进外泌体释放，导致EPCs上miR-21下降，其靶基因WNT1升高，调控EPCs分化；抑制EPCs上miR-21表达，促进EPCs向成骨样细胞表型转化。由此我们假说糖尿病环境下外泌体促进miR-21向细胞外转运降低后者细胞内表达，上调其靶基因导致血管钙化。本研究拟先确立miR-21调控血管钙化的机制和作用，明确外泌体转运miR-21的功能和机制；体内实验证实miR-21在血管钙化中的作用；临床观察研究明确miR-21对预测心血管事件的的应用。本研究将为预防血管钙化和治疗心血管疾病提供新的靶点和理论依据。

血管钙化是老年人和糖尿病患者动脉粥样硬化的重要病理表现；血管钙化增加了血管的硬度，降低了血管的顺应性，显著增加心血管意外的危险。内皮祖细胞(endothelial progenitor cells, EPCs)上基因表达改变和分化异常是血管内膜钙化的主要原因。.本项目研究：（一）成功构建EPCs成骨诱导模型和氧化应激模型，通过转染过表达和干扰miR-21慢病毒至EPCs，发现miR-21对抑制EPCs向成骨样细胞分化。（二）明确糖尿病环境促进外泌体释放，外泌体上MiR-21表达增加。（三）明确miR-21调节EPCs成骨样细胞分化的靶基因为Wnt1，同时对靶基因下游机制的研究。MiR-21通过靶基因Wnt1调控EPCs向成骨样细胞分化。（四）在氧化应激环境下（AGEs刺激下），细胞上清液中外泌体中miR-21表达增加。进一步明确了miR-21通过TGF-β-Wnt-1信号轴和p-P38\p-JNK蛋白磷酸化调控EPCs血管钙化。（五）同时用miR-21-/-小鼠或WT小鼠的骨髓移植APOE-/-小鼠，构建在体血管钙化模型，在体验证miR-21调控动脉粥样硬化作用和机制。（六）在糖尿病患者血浆中MiR-21表达下降，同时和血管钙化相关；EPC-Exos上的miR-21表达与血管钙化积分负相关，是冠心病患者预后的潜在新指标。.该项目首次锁定miR-21和外泌体在血管钙化和动脉粥样硬化中的作用，并揭示miR-21及外泌体对氧化应激环境刺激下细胞转化和成骨相关基因表达发挥关键作用，具有独创性。该项目明确了miR-21及外泌体调控EPCs成骨样分化，改善糖尿病血管内皮损伤的机制，为糖尿病患者血管治疗microRNA、外泌体治疗动脉粥样硬化提供基础研究支撑。.本课题在基金委资助下，完成相关研究实验和预期成果，相关研究共发表本课题第一标注论文7篇（SCI论文6篇），第二标注SCI论文1篇。课题研究期间，授权实用新型专利两项。负责人杨虹波获得上海市“医苑新星”青年医师专科医生人才计划，获第十一期上海市医务职工“星光计划”二等奖和第十期上海市医务职工“星光计划”三等奖等奖项。本课题资助下，指导研究生谭海鹏在Advanced Science（影响因子16.8分）发表SCI论文1篇，该研究生学业优秀，已经顺利转博。..