去乙酰化酶SIRT1的SUMO化修饰与早产儿高氧肺损伤的临床及实验机制研究

The mechanism of preterm hyperoxia lung injury involved in the apoptosis of alveolar epithelial cells (AEC) which induced by P53. After being sumoylated at post-translation level and located in the nucleus, human sirtuin 1 (SIRT1) had deacetylase activity and could inhibit cell apoptosis by deacetylating P53. Our previous study found a seemingly paradox result that SIRT1 increased at the level of protein translation but the deacetylation of P53 decreased in type II AEC after hyperoxia exposure. Literatures reported that the desumoylation of SIRT1 could regulate its deacetylase activity. Thus, we presume that some post-translational modifications of SIRT1 regulate its deacetylase activity in type II AEC after hyperoxia exposure, and the sumoylation of SIRT1 may be the key upstream regulator of cell apoptosis induced by P53 in preterm hyperoxia lung injury. In this study, we will prove our hypothesis at molecular, cell, tissue and population level in preterm newborns, hyperoxia exposure newborn rats and type II AEC, by using gene knockout/silence technology, Western blot, confocal laser scanning microscope and liquid chromatography tandem mass spectrometry (LC-MS/MS). We would like to clarify the role of SIRT1 sumoylation in the mechanism of preterm hyperoxia lung injury, and provide new targets for early intervention and treatment of bronchopulmonary dysplasia.

早产儿高氧肺损伤机制涉及P53介导的肺泡上皮细胞（AEC）凋亡。研究显示，当SIRT1在翻译后水平发生SUMO化修饰并定位于细胞核后获得去乙酰化酶活性，可去乙酰化P53抑制细胞凋亡。但我们对II型AEC的研究却发现似乎矛盾的结果：高氧暴露后细胞内SIRT1增加（翻译水平），但P53去乙酰化减少。另有文献报道，SIRT1翻译后水平的去SUMO化修饰可抑制其酶活性。结合文献和前期研究，我们推测高氧暴露II型AEC中存在调节SIRT1活性的翻译后机制，SIRT1的去SUMO修饰是早产儿高氧肺损伤中P53的上游调节因素。本项目以早产儿、高氧暴露新生动物及细胞模型为研究载体，采用基因敲除/沉默、Western blot、激光共聚焦及液相串联质谱等技术，从人群、组织、细胞及分子水平验证这一假说，阐明SIRT1 SUMO化修饰在早产儿高氧肺损伤发生机制中的作用，为支气管肺发育不良的早期干预提供新的靶点。

支气管肺发育不良（BPD）作为早产儿一种慢性肺疾病，高氧暴露后氧化应激是其发病的关键因素，发育不成熟的肺泡上皮细胞（alveolar epithelial cells, AEC），特别是II型AEC，抗氧化能力低下，常常成为早产儿高氧暴露下肺氧化应激损伤的主要靶细胞。本项目通过建立前瞻早产儿临床队列，以外周血单个核细胞为研究载体，证实了早产儿高氧暴露后诱导ROS产生，导致以 SIRT1翻译后SUMO化修饰调节为中心的SENP-SIRT1-p53信号通路改变与BPD发生发展密切相关，临床上可将上述信号分子作为BPD的早期预测指标；通过构建高氧肺损伤的动物模型和细胞模型，建立SENP基因敲除实验动物模型和SENP基因沉默细胞体系，从整体和细胞水平证实了高氧暴露后通过氧化应激触发SENP1转录活性增加，继而SIRT1蛋白减少、去SUMO修饰增加，出现SIRT1亚细胞定位改变（核-浆穿梭增加）进而影响到p53乙酰化减少，导致肺泡上皮细胞凋亡，是早产儿BPD发病的重要机制；证实了布地奈德、咖啡因、白藜芦醇可能通过上述信号途径对早产儿BPD具有预防或和保护作用；尚未发现SIRT1 SUMO修饰位点改变与BPD发病的关系。该研究从人群、组织、细胞及分子水平阐明了SIRT1 SUMO化修饰导致其乙酰化活性降低是早产儿高氧肺损伤的重要机制，为临床认识BPD的发病机制提供了实验室性依据和采取干预措施防治BPD的发生和发展提供了新的靶点。