NOTCH1基因启动子区甲基化对主动脉瓣钙化的调控机制研究

The incidence of aortic valve calcification (AVC) is increasing year by year. The treatment of AVC is currently limited to operation, still lack of effective preventable measures. Studies show the main mechanism is aortic valve interstitial cells differentiate into osteoblast-like cells and regulated by various factors, which contribute to the study of effective drugs could prevent and treat the progression of AVC. DNA methylation plays an important role in calcification, DNA methylation can alter the methylation levels of promoter CG sites which mediated by methylation transferase, and then inhibit vascular smooth muscle cells differentiate into osteoblast-like cells. The NOTCH1 gene linked to aortic valve calcification, in vivo and vitro studies confirmed that NOTCH1 can inhibit the pathway of osteoblast like cells calcification. On this basis, we intend to establish aortic valve calcification models which contain cell model in vitro and animal model in vivo in this study. This procedure help us to investigate the DNA methylation mechanism of NOTCH1 regulating aortic valve calcification from cell level to organism level, and we initially try to investigate whether demethylation drugs will block this effect. The study of this research will help to reveal the epigenetic mechanism of aortic valve calcification and provide novel idea for the prevention and treatment.

主动脉瓣钙化的发病率逐年增高，目前对其治疗手段仅局限于手术，尚缺乏有效的预防或干预措施。研究表明主动脉瓣钙化发生发展的主要机制是主动脉瓣间质细胞向成骨样细胞分化，同时该过程受各种因素调控，这就使得预防或延缓主动脉瓣钙化的进展成为可能。DNA甲基化在钙化方面发挥了重要作用，在DNA甲基化转移酶的介导下，改变基因启动子区CG位点的甲基化水平，可抑制血管平滑肌细胞向成骨样细胞分化。NOTCH1基因与主动脉瓣钙化相关，体内和体外的实验均证实NOTCH1可抑制成骨样细胞钙化途径。在此基础上，本研究拟建立主动脉瓣钙化的体外细胞和体内动物模型，从细胞到整体水平研究NOTCH1基因的DNA甲基化对主动脉瓣钙化的影响机制，并初步尝试去甲基化药物是否能阻断其作用。本项目的研究，有助于揭示主动脉瓣钙化的表观遗传学机制，为主动脉瓣钙化的防治提供新的思路。

主动脉瓣钙化的发病率逐年增高，目前对其治疗手段仅局限于手术，尚缺乏有效的预防或干预措施。研究表明主动脉瓣钙化发生发展的主要机制是主动脉瓣间质细胞向成骨样细胞分化，同时该过程受各种因素调控，这就使得预防或延缓主动脉瓣钙化的进展成为可能。DNA甲基化在钙化方面发挥了重要作用，在DNA甲基化转移酶的介导下，改变基因启动子区CG位点的甲基化水平，可抑制血管平滑肌细胞向成骨样细胞分化。体内和体外的实验均证实NOTCH1与主动脉瓣钙化显著相关，其机制可抑制成骨样细胞钙化途径。.在本研究中，在CAVD组织中瓣膜间质细胞成骨分化中甲基转移酶的表达上调可以下调的Notch1表达，提示DNA甲基化在Notch1表达和CAVD进展中的潜在作用。在成骨分化诱导的第14天，CAVD组织中Notch1启动子中的甲基化水平比人AVIC中高。 Notch1细胞间结构域（NICD）的沉默促进成骨分化，而去甲基化剂5-Aza-dC的处理抑制了成骨分化。此外，NICD过表达显著抑制了β-catenin对TCF4的转录活性和成骨分化因子的表达，提示Wnt /β-catenin信号参与Notch1调节hAVICs中的成骨分化。总之，Notch1启动子甲基化导致Notch1表达降低，随后随着hAVIC细胞核中NICD的释放减少，从而促进Wnt /β-catenin信号的激活和成骨分化因子的表达，最终促进hAVICs中的成骨分化。DNA甲基化可能是连接表观遗传变异和CAVD进展的重要桥梁。