NGF对心脏内皮细胞Agrin表达的调控及其在心肌再生中的作用

Myocardial regeneration has been studied for more than 20 years. However, full myocardial regeneration is still hard to achieve. Recent studies found that extracellular matrix components Agrin, which is mainly expressed in cardiac endothelial cells, regulated cardiomyocyte proliferation and regeneration by the Yap pathway. However, the mechanism of the regulation of Agrin expression in cardiac endothelial cells is unclear. Our preliminary in vitro study showed that the treatment with exogenous nerve growth factor (NGF) increased Agrin expression in cardiac microvascular endothelial cells (CMECs). Accordingly, we first aim to explore the regulation mechanisms of Agrin expression by NGF in CMECs. Then, we will investigate the role of the NGF-TrkA-CREB-Agrin pathway in cardiomyocyte proliferation, maturation and regeneration by using the TrkA-knockout mice. Our study will increase our knowledge of the specific function of cardiac endothelial cells in myocardial regeneration. An in depth understanding of the NGF-TrkA-CREB-Agrin pathway may help identify new targets to improve myocardial regeneration.

包括细胞移植在内的心肌再生研究已逾20年，但仍缺乏有效手段实现充分心肌再生。最新研究发现主要来源于心脏内皮细胞的细胞外基质成分Agrin能通过Yap途径抑制新生小鼠心肌成熟，促进成年小鼠心肌细胞去分化、增殖，标志着心肌再生研究进入“基质医学”阶段。然而，内皮细胞产生Agrin的上游调控机制尚不清楚。我们前期研究发现外源性NGF能在体外上调心脏微血管内皮细胞(CMEC)Agrin表达。据此，本项目拟在CMEC上通过干预和激活NGF信号途径的不同环节，从细胞层面探讨NGF对CMEC Agrin表达的调控机制；利用 TrkA(NGF受体)心血管内皮细胞特异性基因敲除小鼠，探讨在体模型中心脏内皮细胞NGF-TrkA-Agrin途径对心肌细胞增殖、成熟及损伤后再生的影响，进一步阐明心脏内皮细胞功能对于心肌再生的作用，为以Agrin为靶点的心肌再生治疗策略提供新的思路和依据。

本研究旨在探讨NGF对心脏内皮细胞Agrin表达的调控机制。首先验证了出生1天（P1）、7天（P7）及6周（6W）C57BL/6小鼠心脏内皮细胞Agrin的表达，发现Agrin的表达随着小鼠的年龄增长而显著降低。然后建立了P1、P7及6W C57BL/6小鼠原代心脏内皮细胞的培养技术，通过流式细胞术（FCAS）及免疫荧光检测心脏内皮细胞所占比例均>70%。最后我们通过不同浓度NGF刺激P1、P7小鼠原代心脏内皮细胞，发现在5n/ml NGF能刺激P7小鼠原代心脏内皮细胞Agrin的增加。通过荧光素酶报告基因系统检测了Creb转录因子对Agrin表达实验证实NGF不通过CREB途径上调心脏内皮细胞的Agrin表达。我们将继续通过生物信息学手段探索并验证NGF上调心脏内皮细胞Agrin表达的信号通路，并准备开展NGF对成鼠心梗模型心肌再生动物实验，期望为心肌再生治疗提供新的干预手段。