转录因子Hey在Notch通路调控心肌组织Ⅰ型和Ⅲ型胶原合成异常增多中的作用及其机制研究

Excessive increased syntheses of type Ⅰ and Ⅲ collagens, is one important characteristic of differentiation from cardiac fibroblasts (CFs) into myofibroblasts (MFs), and it is also the primary pathogenesis of myocardial fibrosis (MF). It has been proved that blocking Notch pathway could induce differentiation from CFs into MFs in our previous study, but the molecular mechanisms underlying the excessive increased syntheses of collagens during the process of CFs trans-differentiation regulated by Notch pathway remains unclear. Inhibitory transcriptional factor Hey, regulated by RBP-J, is an important target protein of Notch pathway. Bioinformatics analysis indicates that there are binding sites of Hey at the promoter region of collagen genes. We presume that blocking Notch pathway could decrease the activity of RBP-J, down-regulate the expression of Hey. As a result, the expression of type Ⅰ and Ⅲ collagen genes were up-regulated and the collagen syntheses increased. In this subject, we will focus on the regulatory roles and its mechanisms of Hey on the expression of collagen genes, observe the possibility of Notch pathway acting as the common path of the excessive collagen syntheses in cardiac tissues induced by different profibrogenic factors, discuss the possibility of Hey acting as the key molecular of regulating the expression of type Ⅰ and Ⅲ collagen genes, ascertain the exact binding site of Hey on type Ⅰ and Ⅲ collagen genes. Through hard work, we will provide undoubted evidences and expound the pathological mechanisms of MF in this study.

心肌成纤维细胞(CFs)转分化为肌成纤维细胞(MFs)，导致心肌组织Ⅰ、Ⅲ型胶原合成异常增多，是心肌纤维化(MF)发生的根本原因。我们前期研究证实阻断Notch通路可促进CFs向MFs转分化，但Notch通路如何调控CFs转分化过程中胶原合成异常增多的机制尚不清楚。抑制性转录因子Hey是Notch通路中受RBP-J调控的靶蛋白，生物信息学分析提示Ⅰ、Ⅲ型胶原的启动子区域存在Hey的结合位点。我们推测阻断Notch通路可通过降低RBP-J活性，下调Hey的表达，进而上调Ⅰ、Ⅲ型胶原基因表达，导致胶原合成增多。本课题拟以Hey对Ⅰ、Ⅲ型胶原基因的表达调控为切入点，综合利用各种分子生物学技术，观察不同致MF因素是否均可通过Notch通路诱导胶原合成异常增多，探讨Hey作为调控Ⅰ、Ⅲ型胶原表达的关键分子的可能性，确定Hey与Ⅰ、Ⅲ型胶原基因的确切结合位点，为深入阐明MF的发生机制提供新的实验依据。

心肌纤维化(MF)是心力衰竭和心源性猝死发生的重要病理基础，心肌组织中Ⅰ型和Ⅲ型胶原含量异常增多导致MF，心肌成纤维细胞(CFs)转分化为肌成纤维细胞(MFs)是心肌组织中Ⅰ型和Ⅲ型胶原合成异常增多的根本原因。Notch信号通路是调控多种成体细胞转分化的重要信号通路，但是Notch通路是否参与了CFs向 MFs转分化，目前尚不清楚。本研究分别以大体C57小鼠心肌梗死后MF模型和体外培养的血管紧张素Ⅱ(AngⅡ)诱导的CFs-MFs转分化模型为研究对象，探讨了Notch通路及其下游信号分子Hey在MF中的作用。结果显示：① 在C57小鼠心肌梗死后MF过程中，心肌组织中Ⅰ型和Ⅲ型胶原含量异常增多，同时伴有Notch1及Hey1 mRNA和蛋白表达显著增高，给小鼠心肌梗死后每天腹腔注射Notch通路γ-分泌酶抑制剂DBZ，可使其心肌组织中Notch1蛋白及Hey1 mRNA和蛋白表达均显著下降，同时伴随Ⅰ型胶原合成显著减少；② 在体外培养的AngⅡ诱导的CFs向MFs转分化过程中，Notch1及Hey1 mRNA和蛋白表达也显著增高，使用AngⅡ AT2受体拮抗剂可使CFs Notch1及Hey1 mRNA和蛋白表达均显著下调，同时抑制CFs向MFs转分化，使用Notch1和Hey1 SiRNA转染CFs，同样可以显著抑制Notch1及Hey1 mRNA和蛋白表达，同时抑制CFs-MFs转分化。本研究结果提示：Notch1及其下游信号分子Hey1表达增高可能参与了CFs-MFs转分化，导致心肌组织中胶原含量异常增多，Notch通路激活可能促进了心肌组织病理状态下MF的发生，但是其因果关系还需要进一步大体动物基因功能试验的验证。