ILK 介导的 Tβ4 预处理在心外膜细胞分化为心肌、血管平滑肌细胞中的作用及机制

Recent study demonstrated that in mice pretreated with thymosin beta4 (Tβ4) and subsequently subjected to experimental myocardial infarction (MI), a subset of epicardial cells differentiated into cardiomyocytes, however, the differentiation efficiency is low and regulating mechanisms are still not clear. Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase and a key element in the regulation of cell proliferation、emigration、differentiation、apoptosis and survival, by phosphorylateing the protein kinase B (PKB) and glycogen synthase kinase 3β (GSK3β). Our preliminary studies showed that Wnt/β-Catenin pathway was actived in the differentiation of epicardial cells into vascular smooth muscles. Using epicardium genetic lineage trace line Wt1GFPCre mice, we will investigate the activation of Tβ4 and the involvement of specific ILK pathway proteins in the developing hearts and Tβ4 primed hearts prior to MI. We will investigate the effects and mechanisms of ILK pathway on epicardial cells under different conditions and on the differentiation of epicardial cells into cardiomyocytes and vascular smooth muscle cells. Finally, we will examine whether manipulating ILK pathway can improve the differentiation efficiency of epicardial cells into cardiomyocytes and vascular smooth muscle cells. This study could lead to new strategies for cardiac repair.

最新研究显示 Tβ4预处理的心脏损伤后可激活心外膜细胞分化为心肌、血管平滑肌细胞，但分化率低且调控机制不明。ILK是一种机体内广泛分布的丝/苏氨酸蛋白激酶，通过磷酸化下游底物 PKB和GSK3β(GSK3β磷酸化后激活 Wnt/β-Catenin信号通路)参与信号通路转导，调控着细胞的增殖、迁移、分化、凋亡和存活。前期试验发现胚胎心外膜细胞分化为血管平滑肌细胞过程中，Wnt/β-Catenin 被激活。本项目拟用 Wt1GFPCre 小鼠为试验对象，观察 Tβ4 及 ILK 介导的信号通路关键分子在小鼠心脏发育和Tβ4 预处理的心脏梗死后的表达变化及 Tβ4 和 ILK的关系；观察ILK介导的信号通路体外对不同条件下心外膜细胞生物学行为及分化为心肌、血管平滑肌细胞的影响，并阐明其机制；进一步探讨体内调控 ILK 介导的信号通路能否提高心外膜细胞的分化效能，为临床心肌损伤治疗提供理论基础。

明确心外膜细胞向血管平滑肌细胞、心肌细胞分化的机制与调控因素，为临床心肌损伤治疗提供理论基础。本课题探索了ILK 介导的信号通路在心外膜细胞向血管平滑肌、心肌细胞分化中的作用。结果发现：（1）与对照组相比，Tβ4、ILK 及 ILK 介导的信号通路关键分子在 Tβ4 预处理的心脏受损后表达显著增加，且随着时间的推移，表达增加。（2）无氧无血清条件下，Tβ4 预处理的心外膜细胞能显著的降低凋亡和促进增殖，促进心外膜细胞表达血管平滑肌细胞特异性基因及蛋白，但不表达心肌细胞特异性基因及蛋白，ILK 信号通路关键分子的表达也显著增加；拮抗 ILK 信号通路后，凋亡率显著增加，增殖率显著下降，心外膜细胞向血管平滑肌细胞分化率显著下降。（3）移植 Tβ4 预处理的心外膜细胞到梗死后心脏周边区，能显著的减少梗死周边的细胞凋亡、炎症反应；显著减少心肌纤维化和心室重构，改善心功能；增加心外膜细胞表达血管平滑肌细胞特异性蛋白，和心肌细胞特异性蛋白。拮抗 ILK 信号通路后，梗死周边区的细胞凋亡、炎症反应显著增加，心室增大，心功能恶化，检测不到心外膜细胞表达血管平滑肌细胞特异性蛋白，和心肌细胞特异性蛋白。ILK 介导的信号通路在心外膜细胞分化为心肌、血管平滑肌细胞过程中有着重要作用。