长链非编码RNA-ANCR在动脉钙化中的保护机制研究

Artery calcification is a common problem among the elderly and patients with diabetes or end stage renal disease, and it is an independent risk for cardiovascular events and cerebral infarction. Recent studies suggest that artery calcification is an actively regulated process which is similar to bone formation. The differentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells is considered as the cytology foundation of artery calcification. However, the definite mechanisms have not been expounded. Anti-differentiation non-coding RNA (ANCR), a newly identified long non-coding RNA (lncRNA), has been demonstrated to modulate the process of osteoblast differentiation. Our previous studies showed that ANCR could inhibit the osteoblastic differentiation of VSMCs through inducing autophagy. In this study, we are aimed to construct the adenovirus vector over-expressing ANCR and explore the roles of ANCR in mechanism of artery calcification; both luciferase reporter gene strategies and dominant negative mutant technology will be also used. Then, the vector over-expressing ANCR will be injected into the animal model of artery calcification to evaluate the effects of ANCR at molecular and pathological levels, which will be helpful to demonstrate whether ANCR can be a new target for the clinical therapy of artery calcification.

动脉钙化常见于糖尿病、终末期肾病患者和老年人，是心血管事件、脑梗死发生的独立危险因素。研究表明，动脉钙化是一种类似于骨形成的主动调控过程，血管平滑肌细胞（vascular smooth muscle cells, VSMCs）向成骨样细胞分化是其细胞学基础，然而其具体的发病机制仍不明确。最新的研究表明，一种新发现的长链非编码RNA-ANCR，即抗分化非编码RNA参与调控成骨细胞的分化。我们在预实验中发现，ANCR可能经自噬途径抑制VSMCs的成骨样分化。本项目拟首先建立动脉钙化的细胞模型，构建ANCR过表达腺病毒载体，利用荧光素酶报告基因策略和显性负突变体技术探讨ANCR调控VSMCs钙化的具体机制；然后将ANCR过表达腺病毒载体注入动脉钙化小鼠模型，从分子和病理水平观察ANCR对动脉钙化的影响，明确ANCR能否成为动脉钙化防治的新靶点。

动脉钙化常见于糖尿病、慢性肾脏病和老年人，是心血管事件、脑梗死发生的独立危险因素，动脉钙化是血管平滑肌细胞（VSMCs）向成骨样细胞分化的主动调控过程。近期研究发现，长链非编码RNA-ANCR参与调控成骨细胞的分化。本项目中，我们以β-甘油磷酸钠（β-GP）干预小鼠原代VSMCs构建动脉钙化细胞模型，研究发现在VSMCs向成骨样细胞分化的过程中ANCR的表达降低，以ANCR过表达慢病毒载体转染VSMCs，可显著抑制VSMCs的成骨样分化。该结果提示，ANCR可能是VSMCs成骨样分化的关键分子。进一步的研究发现，ANCR可能通过诱导自噬即促进LC3的表达、削弱NF-κB信号通路抑制VSMCs的成骨样分化。为了进一步探讨ANCR对动脉钙化的影响，课题组以维生素D3干预6周龄C57BL/6 雄性小鼠建立动脉钙化模型，经尾静脉注射ANCR过表达慢病毒载体，分离小鼠胸主动脉，以免疫组化、Von Kossa染色、茜素红染色等方法检测动脉钙化情况及钙化相关指标的表达。研究结果表明，ANCR过表达可显著抑制动脉钙化的发生。ANCR有望成为临床防治动脉钙化的新靶点，本项目的开展为动脉钙化的防治提供了新的思路。