microRNA介导Vaspin调控动脉钙化的机制研究

Artery calcification is a significant risk factor of cardiovascular events, and the osteogenic differentiation of vascular smooth muscle cells(VMSCs) is indentified to be the foundation of onset and development of artery calcification. In the recent studies, they found that the artery calcification level was positively related to the fat mass, and the adipokines played an vital role in this phenomenon. Vaspin was a newly found adipokines, and there is no report of its effect during the artery calcification. In our preliminary experiments, we found that vaspin could promote the osteogenesis of VMSCs, but the mechanismunderlying was unclear. microRNA(miRNA) is an important mechanism in epigenetics modualtion, they widely participate in the regulation of physiological and pathological processes. According to our preliminary study, we assumed that Vaspin could promote the process of artery calcification through the modulation of miRNA. To prove this assumption, the qRT-PCR and western blot will be used to indentify the effect of Vaspin during the osteogenic differentiation of VMSCs, a miRNA chip will be used for miRNA selection; the over-expression and low-expression model will be built to explore the role and target of miRNA; a adviurs vector will be construction for in vivo study. Of this study was to research the role and mechanism of vaspin modulating artery calcification, to fully elaborate the relationship between adipokines and the artery calcification, as well as to provide a theoretical support of how epigenetic mechanisms participate in the prevention and treatment of artery calcification.

动脉钙化是心血管不良事件的重要危险因素，血管平滑肌细胞的成骨样分化是其发生与发展的细胞学基础。近年研究发现脂肪组织含量与动脉钙化水平显著正相关，脂肪因子是其可能的机制之一。Vaspin是一种新的脂肪因子，我们前期研究中发现Vaspin促进血管平滑肌细胞的成骨样分化，但是其具体机制尚不明确。microRNA（miRNA）作为表观遗传学中重要的机制，参与了体内多种病理生理过程的调节。因此在本课题中，结合预实验结果，我们提出假设：Vaspin促进动脉钙化的发生，miRNA是其可能的机制之一。本课题在预实验基础上，将使用miRNA芯片、过表达及低表达模型构建、荧光素酶报告载体、腺病毒转染等技术从体内及体外两个方面对这一假设进行验证。其结果有助于揭示动脉钙化发病的新机制，也为表观遗传学机制在动脉钙化的防治中提供理论依据。

研究背景及目的：动脉钙化是动脉硬化的一种类型,主要发生于大动脉和中动脉管壁的中膜,主要表现为血管壁内小而弥散分布的羟基磷灰石晶体钙沉积。多项大样本调查显示动脉钙化在心血管危险评估中居于重要地位，与心血管不良事件的发生显著相关，但是其发病机制目前尚不明确。既往研究认为动脉钙化是血管平滑肌主动的成骨分化，因此本研究主要研究了脂肪因子Vaspin在血管平滑肌细胞成骨分化中的作用及机制研究，旨在揭示肥胖与动脉钙化之间的关系。.研究方法：通过western、q RT-PCR等检测成骨标志物表达明确Vaspin对血管平滑肌细胞成骨分化的作用，microRNA芯片筛选参与了此过程的miRNA。通过miRNA mimics /inhibitor构建过表达/低表达模型，研究不同miRNA表达水平对Vaspin调控血管平滑肌细胞成骨分化的作用及靶基因。利用免疫组化明确Vaspin对小鼠动脉钙化的影响。.研究结果：1.Vaspin干预血管平滑肌细胞后其碱性磷酸酶（ALP）、骨钙素（OC）、RUNX2等成骨指标均明显升高。2.Vaspin干预血管平滑肌细胞后多个miRNA表达发生了变化，其中miR-466d-3p、miR-7212-3p表达上调，同时miR-328-5p、miR-423-5p、miR-1191b-5p表达下调。3.过表达miR-423-5p可以减弱Vaspin对血管平滑肌细胞成骨分化的促进作用。4.过表达/低表达miR-423-5p可以抑制/促进RUNX2的表达。5.Vaspin过表达促进小鼠胸主动脉钙化的进展。.结论：Vaspin可以促进血管平滑肌的成骨分化，这一作用可能是通过下调miR-423-5p从而促进RUNX2表达实现的。本研究有利于完整阐述肥胖与动脉钙化之间的关系，同时也为表观遗传学机制在动脉钙化的预防及治疗中的应用提供新的理论支持。