GSK-3β调控血管平滑肌细胞特异性转录因子Myocardin对动脉粥样硬化斑块形成作用及分子机制

Atherosclerosis (AS) ，which can cause lots of serious diseases including Coronary heart disease, Cerebrovascular disease and diabetes so on, is the main reason of human death worldwide.The relationship between AS plaque formation and the proliferation and differentiation of Vascular Smooth Muscle Cell (VSMC) is extremely close. Myocardin is a specific transcription factor of VSMC, which is critical inducible factor for smooth muscle marker genes like SM22, Calponin. However, It is still not clear which upstream regulatory factors may affect its myogenic activity in response to extracellular signals and what is the signal transduction mechanism. Based on previous studies and preliminary experiments, The hypothesis of this project is that GSK-3β can positively regulate Myocardin which affect on AS plaque formation. First, we will select recombinant plasmid, and construct VSMCs, which can regulate objective gene expression. Then, we will do GSK-3 β plasmid gene transfection to change the activity of GSK-3β in VSMCs in order to explore the role and mechanism of GSK-3β regulation on the transcription and expression of Myocardin and the influence on mRNA and protein level of SM22，Calponin. For this project, we will improve protein immunoprecipitation and Western blot technology to research the mechanism of the regulation on protein about histone covalent modification. And we will study on quantitative analysis of protein in cell level by using confocal laser scanning microscopy. Moreover，we will combine chromatin immunoprecipitation with qPCR technology to study the specific site and molecular mechanism of transcriptional regulation. On the other hand, We will do the related experiment on the animals developed with AS. In short, our goal is to make sure whether and how GSK-3β can regulate Myocardin, the specific transcription factor in VSMC which affect on AS plaque formation. If so, we can find target gene and the effective drug for the prevention and treatment on Atherosclerosis.

动脉粥样硬化(AS)与血管平滑肌细胞（VSMC）的增殖和分化密切相关。Myocardin是VSMC特异性转录因子，是VSMC 标志基因的关键诱导因子，针对Myocardin生肌活性调控因子及机制未知，在前期研究和预实验基础上，提出GSK-3β通过组蛋白共价修饰来正性调控Myocardin的转录水平与活性的假说。拟采用基因重组质粒，构建灵敏的可调控目的基因表达的VSMC系，通过基因转染及药物调控GSK-3β活性，研究对Myocardin转录与表达作用及对下游基因mRNA与蛋白水平的影响。采用蛋白免疫共沉淀结合Western blot改良技术研究组蛋白共价修饰的调控机制，并进行蛋白的定量分析研究；采用染色体免疫共沉淀结合qPCR关键技术研究转录水平调控的具体位点及分子机制；同时开展相关动物实验。旨在明确GSK-3β对Myocardin的调控作用及分子机制，为AS防治提供理论依据及分子靶点。

动脉粥样硬化（AS）是导致心血管疾病的主要病理基础,血管平滑肌细胞(VSMCs)的增殖和分化在AS疾病进程中起到非常重要的作用，而VSMCs 的特异性转录因子Myocardin是VSMCs标志基因的关键诱导因子。针对Myocardin 的生肌活性调控因子及其信号机制未知，课题组构建了稳定的实验细胞系，应用基因重组质粒转染、免疫共沉淀法、Real Time Q-PCR、Western Blot等分子生物学技术进行血管平滑肌细胞实验和AS动物模型体内实验，系统研究GSK-3β调控Myocardin的分子机制及对AS进程的影响。我们发现：在血管平滑肌细胞中GSK-3β抑制剂能降低Myocardin中丝氨酸（S57-469，S627-639）和苏氨酸位点的磷酸化，可减少Myocardin对其靶基因ACTA2启动子的募集；从而降低Myocardin靶基因及基因产物CNN1 (calponin)、TAGLN1 (SM22)、ACTA2 (SMα-actin)的表达；显性失活型GSK-3β突变体的高表达能模拟GSK-3β抑制剂的效应；持续活性型和野生型GSK-3β的高表达可增加calponin、SM22和SMα-actin的表达。在AS动物体内实验中，由于机体功能调控的复杂性，存在多通路的相互作用， AS组的NF-κB上调，影响Myocardin，下调其靶基因SM22的转录表达；而当用GSK-3β抑制剂处理后，NF-κB及其相关炎性因子下调，可缓解AS疾病进程。本课题研究结果丰富了人们对血管平滑肌细胞(VSMCs)的增殖和分化在AS斑块形成作用机制的认识，并可为防治AS性疾病提供新的思路。