调控核小体装配蛋白类似蛋白1（NAP1L1）抑制缺血再灌注心肌凋亡的机制研究

Nucleosome assembly protein 1-like 1(NAP1L1) promotes cell proliferation by regulation of DNA replication. However, whether it plays a role in cardiovascular system is unclear. Recently, we observed cardiac-specific overexpression of NAP1L1 inhibits cardiomyocyte apoptosis induced by ischemic-reperfusion (I/R) injury, cardiac-specific LRP6 overexpression enhances nuclear NAP1L1 expression , serum WNT2, one of ligands of LRP6, is increased in patients with acute myocardial infarction. On the basis of these data, we will build I/R model with cardiac specific-NAP1L1-overpression rat, tamoxifen inducible cardiac specific-LRP6-overexpression or downregulation mice, inject cardiomyocyte specific WNT2-AAV9 and/or active GSK3β-AAV9 to observe cardiac function, cardiomyocyte apoptosis and the expression of LRP6, NAP1L1 and GSK3β et al. In vitro, we will culture cardiomyocytes and respectively transfected lenti-WNT2, lenti-LRP6, lenti-sh-LRP6, lenti-NAP1L1, lenti -GSK3β (active) to observe apoptosis induced by H2O2. We will clarify the molecular mechanisms that WNT2/LRP6 inhibits the activation of GSK3β and then enhances NAP1L1 expression or its translocation to nucleus to protect heart from apoptosis against I/R. It will be of great significance to prevent cardiac injury induced by I/R.

核小体装配蛋白类似蛋白1（NAP1L1）主要调控DNA复制促进细胞增殖，但它对心肌的作用有待阐明。我们在最近观察到心肌特异性过表达NAP1L1可抑制缺血再灌注（I/R）心肌凋亡、心肌过表达WNT受体LRP6可促进I/R心肌NAP1L1入核并抑制心肌凋亡、急性心梗初期病人血清WNT2上调等基础上，拟通过心肌特异性NAP1L1过表达大鼠及诱导型心肌特异性LRP6过表达或下调小鼠建立I/R模型，注射心肌特异性WNT2和/或激活型GSK3β腺相关病毒，研究WNT2激活LRP6受体抑制GSK3β活性促进NAP1L1表达或入核来抑制I/R介导的心肌凋亡从而发挥心脏保护作用的具体分子机制。并通过培养的心肌细胞分别转染WNT2及sh-LRP6等有关慢病毒，用过氧化氢介导氧化应激，观察心肌凋亡以明确以上机制。本研究将初步阐明NAP1L1保护I/R心肌及其上游调控机制，有利于发现新靶点防治I/R心肌损伤。

如何减轻心脏再灌注损伤已成为医学界的研究热点之一。在本研究中，我们观察到在小鼠心脏缺血再灌注损伤模型中，心脏缺血区组织和血清中WNT2水平下降，在加入外源性的重组WNT2蛋白后显著改善缺血再灌注损伤心脏心功能。进一步研究发现WNT2通过心肌细胞膜受体LRP6促进NAP1L1的泛素化降低NAP1L1水平，从而抑制缺血再灌注损伤心肌凋亡及铁死亡等发挥保护作用。此外，WNT2和WNT4通过LRP6与FZD4/2的协同作用调控心梗后的纤维化。我们还发现了心肌LRP6促进HSF1入核介导GSK3β和AMPK磷酸化抑制缺血再灌注心肌凋亡，以及促进WNT5a和WNT11降解抑制压力负荷诱导的心肌纤维化作用。这些发现可为缺血介导的心肌损伤的有效干预提供了新的治疗靶点。共发表该基金标注的SCI论文5篇。相关研究成果已被国内外学术会议多次交流，并培养博士2名，硕士5名，已毕业3名硕士。