Twist1在血管平滑肌细胞增殖和肺动脉高压肺血管重塑中的作用及机制研究

Pulmonary hypertension (PH) is a severe pulmonary vascular disease that is associated with extremely high mortality. Deeper understanding of its biological mechanism may provide novel strategies for clinical therapy. Previously, we have observed significantly elevated expression of Twist1 protein in the remodeled pulmonary arteries and hypoxia-treated pulmonary arterial smooth muscle cells. Furthermore, knockdown of Twist1 in pulmonary arterial smooth muscle cells significantly inhibited cell proliferation and migration. Our previous findings suggests that DDX5 plays an important role in vascular remodeling and smooth muscle cell proliferation, and our recent study has shown that Twist1 might be a negative regulator of DDX5 signaling. In combination with the recent progresses in the field of PH, we hypothesized that Twist1 might aberrantly suppressed DDX5/miR-204 pathway, and played an important role in pulmonary arterial smooth muscle cell proliferation and migration, which further contributed to vascular remodeling. In this project, we planned to first evaluate the transcription and expression levels of Twist1 and DDX5/miR-204 signaling using RT-PCR and immunofluorescence; analysis. Second, we would establish Twist1 knock-out mice, hypoxic pulmonary hypertension animal model, Twist1 knockdown and overexpression vascular smooth muscle cells, and further study the role of Twist1 in vascular remodeling and smooth muscle cell proliferation and migration. The results might bring new targets for clinical therapy for PH.

肺动脉高压是致死率极高的恶性肺血管疾病，深入研究其发生、发展的生物学机制将为其临床诊治提供新思路。在前期工作中我们发现：重要的转录因子Twist1在重塑肺血管和低氧刺激的肺血管平滑肌细胞（VSMC）中呈异常高表达，而敲低Twist1显著抑制肺VSMC增殖、迁移，提示Twist1在肺动脉高压肺血管重塑中很可能具有重要的调控作用；结合Twist1信号通路及肺血管变构的分子机制，我们推测：Twist1可能通过调控DDX5/miR-204信号途径参与肺动脉高压肺血管重塑进程。本课题组拟通过RT-PCR等方法检测重塑肺血管和低氧刺激肺VSMC中Twist1通路分子的转录、表达；构建Twist1血管条件性基因敲除小鼠肺动脉高压模型及Twist1过表达和低表达VSMC体系，明确Twist1在肺动脉高压进程中的调控作用及具体机制。研究结果有望进一步揭示肺血管重塑的分子机制，为肺动脉高压临床诊疗提供新靶点。

twist1在多种疾病进程中发挥重要的调控细作用，近年来，twist1 作为重要的表观遗传学基因，在肺动脉高压和内皮间充质化转换中起关键作用。然而，twist1在平滑肌细胞中的作用至今尚未研究。本研究从这一关键点出发，来阐述twist1在肺动脉高压进程中在平滑肌细胞中的调控。.本研究通过构建基因条敲小鼠，AAV-病毒特异性敲除大鼠基因，质谱，免疫共沉淀等技术研究平滑肌细胞twist1在肺动脉高压中的作用。.本研究显示，在肺动脉高压病人和大鼠模型中，twist1的表达在显著升高，主要表现在平滑肌细胞中。特异性敲除或降低平滑肌细胞中的twist1的表达可以抑制肺动脉高压的进程在小鼠缺氧和大鼠MCT模型中。体外模型研究显示，抑制twist1或敲低平滑肌细胞中的twist1可以抑制平滑肌细胞的增殖和迁移。.进一步研究显示，twist1主要通过降低bmpr2与gata6的启动子区的结合来发挥调控作用。抑制twist1可以促进bmpr2与gata6启动子区的结合，以恢复bmpr2的调控功能。.本研究探索了平滑肌细胞的twist1在肺血管疾病和肺高压进程中的重要作用，为临床治疗提供新的靶点。