广西壮族人群LPA基因变异与冠心病的相关性及apo(a)下调人THP-1巨噬细胞自噬的机制研究

Lipoprotein a is an independent risk factor of atherosclerosis. The size and concentration levels of lipoprotein a are decided by apo(a). Apo(a) strongly stimulating macrophages to release inflammatory factors may down regulate the autophagy of macrophages, which may be an important factor for the instability of atherosclerotic plaque inducing coronary artery disease. Apo(a) is encoded by LPA gene and is under strong genetic control (heritability=0.68–0.98). Our preliminary study highly indicated that variations of LPA gene rs10455872 and rs3798220 may be the important targets involving in the regulation of peripheral blood lipoprotein a concentration. We will further investigate the correlation between the two polymorphisms of LPA gene and coronary artery disease in Guangxi Zhuang population. The present study includes two parts: Part 1. The correlation analysis of LPA gene rs10455872 and rs3798220 variations with the susceptibility to coronary artery disease, left ventricular ejection fraction and cardiovascular events in Guangxi Zhuang population. Part 2. Discussion on different expression levels of autophagy gene Beclin-1 and LC3-II in human THP-1 derived macrophages in different conditions such as LPA gene heterozygote and homozygote donors derived different apo(a) and the possible mechanism of down-regulation of autophagy of human THP-1 derived macrophages through PI3K-Akt-mTOR pathway.

脂蛋白a是动脉粥样硬化形成与进展的高危因素，脂蛋白a大小和浓度由apo(a)决定。apo(a)具有激活巨噬细胞释放炎症介质的作用，可能通过下调巨噬细胞自噬导致动脉粥样硬化斑块不稳定，参与冠心病发生发展。apo(a)由LPA基因编码，遗传率高达0.68-0.98。我们前期研究发现，LPA基因rs10455872和rs3798220变异可能是影响脂蛋白a浓度的重要靶点。我们拟进一步探讨LPA基因变异与广西壮族人冠心病发生与预后的关系。研究包括：1. 分析LPA基因rs10455872和rs3798220变异与广西壮族人脂蛋白a、冠心病易感性、左室射血分数、心血管终点事件的关系。2. 观察LPA基因杂合子和纯合子供体apo(a)对THP-1巨噬细胞自噬基因Beclin-1和LC3-II表达及自噬体形成的影响，并通过使用PI3K-Akt-mTOR通路抑制剂，探讨apo(a)下调巨噬细胞自噬的机制。

脂蛋白a是动脉粥样硬化形成与进展的高危因素，被公认为冠心病尤其是心肌梗死的高危预测因子。apo(a)是脂蛋白a的致病性结构，可能通过促进炎症介质如IL-8等以及对巨噬细胞自噬的调节导致动脉粥样硬化斑块失稳，进而参与冠心病的发生发展。脂蛋白a浓度不受年龄、性别、BMI、饮食和调脂药物影响，仅与apo(a)有关，而apo(a)几乎仅受遗传调控。通过本项目实施要弄清三个问题：一是广西壮族冠心病脂蛋白a浓度诊断切点在哪里？二是表达apo(a)的LPA基因rs10455872 (A>G)和rs3798220 (T>C)变异与脂蛋白a浓度变化的关系；三是脂蛋白a、apo(a)和氧化型脂蛋白a如何影响巨噬细胞基础性自噬？具体机制是什么？主要结果：■外周血脂蛋白a浓度与广西壮族冠心病发生率呈显著正相关（OR=1.067，P＜0.001），是广西冠心病发生的独立危险因素。广西壮族冠心病脂蛋白a诊断切点为41.44mg/dL，灵敏度和特异度分别为72.4%和79.5%，Youden指数0.5192，曲线下面积0.839。■ LPA 基因 rs10455872和 rs3798220 基因型及等位基因频率的分布在广西壮族冠心病组与正常对照组间差异具有统计学意义。Logistic 回归分析提示LPA基因rs10455872和rs3798220变异彼此独立地与升高的脂蛋白a浓度显著相关。冠心病组脂蛋白a浓度更高，携带更多的TC+CC和AG+GG基因型和更多的C和G等位基因，差异有统计学意义。■脂蛋白a与冠心病严重程度呈显著正相关，与左室功能呈显著负相关，提示脂蛋白a是冠心病进展高危因子。■ mRFP-GFP-LC3双荧光追踪检测及透射电镜观察结果显示，脂蛋白a 、apo(a)干预后THP-1巨噬细胞自噬水平下降，而氧化型脂蛋白a干预后THP-1巨噬细胞自噬水平上升。■ 实时荧光定量PCR结合Western blotting结果显示，PI3K-Akt-mTOR通路对自噬具有双重调节作用：氧化型脂蛋白a通过促进该通路关键靶点mRNA的转录激活巨噬细胞基础性自噬（转录水平）；而脂蛋白a和apo(a)则通过抑制该通路关键靶点蛋白质的降解抑制巨噬细胞基础性自噬（翻译水平）。与氧化型脂蛋白a比较，脂蛋白a和apo(a)抑制巨噬细胞自噬的机制可能更为复杂，如激活ROS通路等。