C5aR1基因新的错义突变与冠心病的关联分析及功能研究

Coronary Artery disease is a multifactorial chronic complex disease, Disorders of blood lipids, blood glucose metabolism and chronic inflammation involved in its pathogenesis. Our previous studies showed that C5L2 involved in blood glucose, lipid metabolism and inflammatory response, play important roles in the development of coronary artery disease. Our previous studies have shown that the genetic polymorphisms of C5L2 were associated with the susceptibility of coronary artery disease and diabetes in Xinjiang different ethnics population. Both C5aR1 and C5L2 were receptor of ASP. In our previous study, we identified a new missense mutation of this gene, but whether this mutation is involved in the development of coronary artery disease and the related mechanism is not clear. In this study, we use TaqMan genotyping technique to analysis the the relationship between the new mutation of C5aR1 gene and the coronary artery disease in Xinjiang Uygur and Han population and the interaction between genes and the environment. Then, at the cellular level, gene knockout mouse model to study the function of the new mutationof C5aR1 gene and further analyze the effect of C5aR1 gene functional on the prognosis of coronary artery disease. To elucidated the new function of C5aR1 gene and its interaction with upstream and downstream genes and proteins, which provided new research for the pathogenesis of coronary artery disease. Reveal the role of C5aR1 gene in the heterogeneity of coronary artery disease in different populations in Xinjiang and the possible molecular mechanism to provide basis for individual diagnosis and the effect of gene mutation on the prognosis of coronary artery disease.

冠心病是一种多因素导致的慢性复杂性疾病，血脂、血糖代谢紊乱及慢性炎症等参与其发病机制。前期研究表明，参与血糖、血脂代谢以及炎症反应的C5L2在冠心病的发生发展中起着重要的作用。C5L2基因多态性和新疆不同民族冠心病、糖尿病的易感性存在关联。C5aR1与C5L2均是ASP的受体，两者有38%的同源性，在动脉粥样硬化斑块中呈现高表达。前期研究鉴定出该基因新的错义突变位点，但该位点是否参与冠心病的发生发展以及相关机制尚不明确。本课题拟在前期研究的基础上利用高通量TaqMan基因分型技术分析C5aR1基因新的变异位点和新疆维、汉民族冠心病的关系及与环境的交互作用；在细胞水平、基因敲除小鼠模型研究C5aR1基因新的突变位点的功能，并分析C5aR1基因功能变异对冠心病预后的影响。初步阐明C5aR1基因新变异的功能及其与上下游基因、蛋白之间的相互作用，为个体化诊断和基因突后对疾病预后的影响提供依据。

本项目在前期研究的基础上，利用高通量TaqMan基因分型技术分析C5aR1基因多态性和新疆不同民族冠心病的关系及其差异;然后在细胞模型及组织标本中验证其功能。整个研究过程分为三个部分，第一部分是基于人群的病例对照研究，分析新的突变位点和新疆维汉民族冠心病的关联。第二部分在细胞水平验证其功能。是第三部分是在动物水平验证其功能。研究结果如下: . 在人群水平，采用病例-对照研究的方法，选择经冠脉造影检查确诊的冠心病患者576例，对照组452例，采用实时荧光定量PCR方法对C5aR1基因新的突变位点进行基因分型，我们发现4个C5aR1基因新的突变位点为罕见突变，测序未能测出。在293T细胞上进行突变位点机制研究，我们发现相比于野生型，C5aR-W213L突变型蛋白表达、mRNA表达和sICAM-1表达含量明显减少，亚细胞定位发生改变。. 细胞实验发现，通过给予THP-1悬浮细胞过表达慢病毒C5aR-WT和C5aR-W213L进行转染，我们发现相比于野生型，C5aR-W213L可减少蛋白、mRNA以及炎症指标的表达，4D非标记定量蛋白质组学通过主要介导新陈代谢、粘附连接、IL-17、补体和凝血级联、胆固醇代谢信号通路等途径进一步发挥作用。. 动物实验发现，通过培育C5aR基因敲除小鼠（C5aR-/-）以及野生型（C57BL6小鼠高脂喂养）,我们发现C5aR信号对小鼠动脉粥样硬化的形成具有损害作用。与C57BL6小鼠相比，高脂饮食喂养24周的C5aR-/-小鼠动脉粥样硬化减轻：如小鼠主动脉和主动脉根部脂质条纹降低；主动脉及全身炎症细胞因子分泌减少；外周血以及主动脉病变处单核/巨噬细胞浸润减少，并趋向于M2型（表达较高水平的CD206,较低水平iNOS）。. 依托此研究基金，项目执行期间申请人作为通讯作者发表基金标注论著12篇，其中SCI论著12篇。课题实施期间培养博士后1名，博士研究生1名，硕士研究生1名。