GAS6/TAM-AMPK信号通路在心脏组织工程瓣膜种子细胞抗流体剪应力损伤中作用及机制研究

Tissue engineering heart valves (TEHVs) have attracted increasing attention with various advantages. Bone marrow mesenchymal stem cells (BMSCs) is widely used as seed cells for TEHVs. However, fluid shear stress injury seriously shortens its lifespan. Nature reported that growth arrest-specific protein 6 (GAS6) takes part in anti-apoptosis, proliferation promotion and anti-inflammation, via binding with its TAM receptors (Tyro3, Axl and Mer) family. We previously confirmed that melatonin protects BMSCs against FSSI through the activation of AMP-activated protein kinase (AMPK) signaling. We further found that ① GAS6 was down-regulated by fluid shear stress injury in BMSCs; ② Knockout of GAS6 decreased the phosphorylation level of AMPK, and aggravated the FSSI in BMSCs; ③ Melatonin protected against FSSI in BMSCs, accompanying the up-regulation of GAS6. Collectively, GAS6/TAM signaling may play a vital role in the protection of BMSCs against fluid shear stress injury. In this project, we will employ fluid shear stress injury model in rat BMSCs to explore the role of GAS6/TAM signaling in the protection of BMSCs against fluid shear stress injury, and the its upstream and downstream relationship with AMPK signaling. Then, we further observe GAS6/TAM-AMPK signaling in the protective effect of melatonin. This project will draw new molecule targets of anti-fluid shear stress injury in TEHVs seed cells, and provide sufficient theoretical basis for the further development of melatonin-based valve-protective treatment.

生物组织工程瓣膜具有生物活性强、血流动力学稳定等优点，成为当前研究热点。骨髓间充质干细胞（BMSCs）是其常用种子细胞，但流体剪应力损伤严重限制BMSCs的寿命。Nature文章指出：GAS6是细胞命运的重要决定者，通过其受体TAM家族发挥抗凋亡、促增殖等作用。我们证实褪黑素可通过激活AMPK通路发挥抗BMSCs流体剪应力损伤作用，还发现：①剪应力损伤后BMSCs中GAS6表达下调；②GAS6敲除后加重BMSCs剪应力损伤，并伴随AMPK磷酸化水平下降；③褪黑素可减轻BMSCs的剪应力损伤，同时上调GAS6表达。以上证据强烈提示：GAS6/TAM通路在BMSCs抗剪应力损伤中扮演重要角色。本项目拟采用大鼠BMSCs剪应力损伤模型，研究证实GAS6/TAM信号通路在抗剪应力损伤中的作用及其与AMPK通路的上下游关系，同时关注该通路在褪黑素瓣膜保护中的作用，从而寻找全新的瓣膜种子细胞保护靶点。

人工瓣膜置换术是治疗严重心脏瓣膜疾病的重要手段，但其寿命短和需终生服用药物抗凝的缺点限制了其临床应用。生物组织工程瓣膜是当前的研究热点，具有生物活性强，使用寿命长，且血流动力学稳定等优点。然而，在心脏血流作用下，作为种子细胞的骨髓间充质干细胞（BMSCs）易发生凋亡和脱落，出现流体剪应力损伤，严重限制了生物组织工程瓣膜的发展。目前防治手段主要包括使用外源性的机械生长因子E肽抗体、血管生长因子等，但这些手段存在价格昂贵、副作用、作用机制不清和伦理学等诸多问题，因此寻找人体内源性生物组织工程瓣膜种子细胞保护物质成为当前研究热点。本研究成功构建了大鼠BMSCs流体剪应力损伤（fluid shear stress injury，FSSI）模型，采用了基因敲除、免疫荧光、Western blot、qRT-PCR等实验方法，发现FSSI能显著降低BMSCs的活率，增加细胞凋亡率和胞内ROS水平，下调GAS6、Axl和AMPK的表达，同时上调Mertk、Tyro3、ACC、Ac-p53、Caspase-3和Bax的蛋白水平。而敲除GAS6能进一步加重FSSI对BMSCs的损伤。褪黑素可上调GAS6、Axl和AMPK的表达，增加细胞活率，减少胞内ROS的积累，减轻BMSCs的剪应力损伤。本研究证实了褪黑素可以激活GAS6/TAM-AMPK信号通路发挥抗FSSI，为开发针对GAS6/TAM-AMPK信号通路来治疗严重心脏瓣膜疾病的方法或药物（褪黑素）奠定理论基础。