新型mTOR抑制分子Deptor在未成熟心肌保护中的作用及机制研究

The exploration of immature myocardial protection has become an urgent task following the development of cardiovascular surgery technology and more younger patients getting the opportunities to regain their health in recent years. Previous studies have found that autophagy can repair the ischemia damage cells. We have already investigated that Spermine could ameliorate ischemia/reperfusion injury in cardiomyocytes via regulation of autophagy and inhibition mTOR signal pathway. However, the specific mechanism of mTOR downstream is still unknown. Deptor is the only direct inhibitory factor for downstream of PI3K/AKT/mTOR signaling pathway, which could also inhibit both mTORC1 / C2 and induce autophagy. Our previous study indicated that in the ischemia/reperfusion injury of immature myocardial cells in vitro, the expression of Deptor significantly decreased and C2 / mTORC1 pathway was activated. Transfection with Deptor could protect the myocardial cells from ischemia-reperfusion injury via suppressing mTORC1 / C2 and promoting autophagy. Based on previous studies, this project wants to verify the hypothesis that Deptor could attenuate the ischemia/reperfusion injury in immature cardiomyocytes in vivo and in vitro. With the help of lentivirus transfection, transgenic animal and electron microscopy immunofluorescence techniques, we want to explore the role of autophagy in the protection of Deptor and the mechanism of mTOR in the regulation of autophagy. The purpose of our study is to find a new target to protect immature myocardium from ischemia injury.

婴幼儿手术日益增多，针对未成熟心肌保护的研究刻不容缓。自噬可以保护缺血再灌注引起的心肌细胞受损，课题组前期研究发现精胺可以抑制mTOR通路，从而诱导自噬，发挥对未成熟心肌细胞的保护作用，但mTOR下游机制不清。Deptor是目前发现唯一的PI3K/AKT/mTOR信号下游直接抑制因子，可以同时抑制mTORC1/2并诱导自噬。我们初步研究发现在体外模拟缺血再灌注损伤培养的未成熟心肌细胞中，Deptor表达明显下降，mTORC1/C2通路激活；上调Deptor表达可以抑制mTORC1/C2，促进自噬发生并减缓缺血缺氧引起的心肌细胞损伤。本研究拟在此基础上，通过CRISPR-Cas9和转基因动物等技术，在体内外心肌细胞损伤模型中验证Deptor在未成熟心肌损伤中的作用，明确自噬在这一过程中的作用，揭示mTOR调控未成熟心肌细胞发生自噬的机制。本研究结果可望为未成熟心肌保护提供新的治疗靶点。

在国家自然科学基金经费资助下，课题组基本按原课题计划书顺利的完成了本项目研究。首先我们通过Deptor Floxp3小鼠与myh6 Cre小鼠杂交构建心肌细胞Deptor特异性敲除的小鼠，并建立了心肌缺血再灌注模型。体内实验结果显示Deptor条件敲除小鼠可以显著增加心肌梗死面积，增加心肌细胞凋亡，而且ELISA实验结果显示CK-MB、LDH和CtnT/I的表达也显著高于同型组，这些结果证明Deptor条件敲除小鼠可以加重缺血再灌注的心肌。同样体外模型我们使用了原代小鼠的心肌细细胞和人的心肌细胞。在 I/R诱导后发现，Deptor siRNA+I/R组可以显著的减少细胞活性和增加心肌细胞凋亡。最后我们对Deptor加重I/R损伤的机制做了进一步研究。结果发现I/R诱导后，Deptor敲除可以显著上调p-mTOR，p-4EBP1,p62的表达，下调LC3的表达。免疫荧光结果显示Deptor敲除后 LC3自噬双荧光减弱，而Deptor质粒作用后，LC3自噬双荧光增强。当使用mTOR C1和C2的共同抑制剂OSI027与Deptor敲除小鼠或者Deptor敲除的人心肌细胞共同作用，体内结果发现梗死面积在Deptor敲除小鼠+OSI027显著减少，而且凋亡的心肌细胞也显著减少，CK-MB、LDH和CtnT/I等酶变化也都显著下调。体外CCK-8检测的细胞活性增强，凋亡细胞减少。这一结果发现OSI027对敲除的I/R模型的小鼠以及I/R诱导的心肌细胞都具有保护作用。综上所述，这些结果证明Deptor可以通过抑制mTOR通路和增加自噬保护I/R诱导的心肌。实验数据已整理成稿，拟投Circulation Research。