Notch信号与TGF-β1/Smad3交互作用调控心肌纤维化的机制研究

Notch signaling pathway has been demonstrated of potent myocardial protection, however, its function in the field of myocardial fibrosis is not well studied. Based on our preliminary work, we will focus on the Notch signaling pathway to identify possible key molecules that regulate myocardial fibrosis in cardiac fibroblasts-myofibroblast transformation induced by TGF-β1.Transgenic technology will be applied to investigate the biological effects of Notch signaling in myocardial fibrosis in many aspects such as cell proliferation, migration, adhesion, collagen synthesis. Interactions between Notch and TGF-β1/Smad3 will be confirmed by co-immunoprecipitation and confocal microscopy, which will illustrate the significance of Notch-TGF-β1/Smad3 feedback mechanism in the regulation of myocardial fibrosis. The regulatory role of interactions between Notch and TGF-β1/Smad3 for myofibroblast marker genes α-SMA will be identified by dual-luciferase reporter assay, quantitative chromatin immunoprecipitation and electrophoretic mobility shift assay, which will elucidate the molecular regulational mechanism of Notch signaling on myocardial fibrosis. Finally, the results will be confirmed in rat myocardial infarction model. This project aims to clarify the mechanism and significance of Notch1 signaling in myocardial fibrosis, and provide a novel target for treatment of myocardial fibrosis.

Notch信号通路具有良好的心肌保护效应，但在心肌纤维化领域尚未深入研究。在前期工作基础上，我们将围绕Notch信号通路，利用TGF-β1诱导心脏成纤维细胞-肌成纤维细胞转化，从中筛选可能调控心肌纤维化的关键分子。再以此为切入点，采用转基因技术，从细胞增殖、迁移、黏附、胶原合成等角度观察Notch信号通路在心肌纤维化中的生物学效应；应用免疫共沉淀及激光共聚焦显微镜探索Notch与TGF-β1/Smad3之间的交互作用，以阐述Notch-TGF-β1/Smad3循环反馈机制在心肌纤维化中的调控意义；利用双荧光素酶报告基因、染色体共沉淀及凝胶迁移技术探讨Notch-TGF-β1/Smad3交互作用对肌成纤维细胞标志性基因α-SMA的表达调节，以阐明Notch调控心肌纤维化的分子机理；最后在大鼠心肌梗死模型上进一步比较论证，力求确定心肌纤维化防治新靶点，为开发新型抗心肌纤维化药物提供理论依据。

我们以Vimentin、DDR2、α-SMA、Tensin表达变化确认TGF-β1诱导CMT成功，从中筛选出Notch1在心肌纤维化中可能具有关键调控作用；以腺病毒载体操纵Notch1在CFB内的表达，发现Notch1通过抑制CMT，减轻CFB的增殖、迁移、黏附、胶原合成能力；Notch1-TGF-β1补偿实验证明Notch1可以废除TGF-β1诱发的心肌纤维化；Co-IP及Confocal microscopy细胞核共定位确认N1ICD与TGF-β1下游分子Smad3存在相互作用，Dual-luciferase reporter assay表明Notch-TGF-β1/Smad3交互作用抑制α-SMA转录表达；建立Sprague-Dawley 大鼠MI模型，从心肌病理变化、羟脯氨酸含量、左心功能角度，进一步比较论证Notch信号通路在心肌纤维化中的调控作用。我们研究表明Notch1信号通路通过对抗Notch-TGF-β1/Smad3介导的CMT，抑制心梗后心肌纤维化；已阐明Notch信号与TGF-β1/Smad3交互作用调控心肌纤维化的相关分子机制，为开发新型抗心肌纤维化药物奠定的理论与实验基础。