lncRNA介导的SIRT1信号网络调控的Ⅱ型肺泡上皮细胞衰老在慢性阻塞性肺疾病中的作用机制研究

The key to treat chronic obstructive pulmonary disease (COPD) is how to repair damaged lung tissue and regenerate the lost alveoli. As the progenitors of type I alveolar epithelial cells, type II alveolar epithelial cells (AECII) play important roles in the maintenance of alveoli structure and function.Our previous study indicated that amniotic fluid-derived mesenchymal stem cells (AF-MSC) can differentiate into AECII in the lungs of the rats with emphysema, and then repair the damaged alveoli. However,there are many problems including in the purity and tumor risk in the differentiation that have not been solved. There are still many difficulties in treating COPD using exogenous stem cells. Therefore, it is useful to study how to repair the damaged lung tissue by the recovery of endogenous stem cells. Premature cellular senescence directly affects the lung progenitor cell function, causing the depletion of stem cells and lung tissue destruction. SIRT1 signaling plays a crucial role in cellular senescence. Long non-coding RNA (lncRNA) directly plays important regulatory roles in the course.Thus, in the present study, lncRNA chip, eukaryotic expression vector transfection, and gene knockout technology will be used to study the mechanisms of type II alveolar epithelial cell senescence regulated by lncRNA-mediated SIRT1 signaling networks in chronic obstructive pulmonary disease and reveal the roles of cellular senescence in the progression of COPD, which will provide new therapeutic targets for COPD.

修复损伤的肺组织、使丢失的肺泡再生是慢性阻塞性肺疾病(COPD)治疗中的关键所在。II型肺泡上皮细胞(AECII)是I型肺泡上皮细胞的祖细胞，在维持肺泡正常结构和功能中起重要作用。我们前期研究发现羊水间充质干细胞(AF-MSC)在肺气肿大鼠肺组织内定向分化为AECII、使肺泡数量增加。但其分化纯度及致瘤风险等问题仍未解决，外源性干细胞治疗COPD仍面临重重困难。因此，恢复内源性干细胞功能以促进肺组织修复值得进一步研究。过早细胞衰老直接影响肺部祖细胞功能，导致干细胞池耗竭及肺组织损伤。SIRT1信号通路在细胞衰老中起着至关重要作用，长非编码RNA（lncRNA）参与细胞衰老调控过程。因此，本项目将应用lncRNA芯片、真核表达载体转染、基因敲除等技术研究肺部关键性lncRNA介导的SIRT1信号网络在AECII衰老中的调控作用，揭示促进COPD进展的细胞衰老机制，为COPD治疗提供新的靶点。

II型肺泡上皮细胞(AECII)数量与功能稳定对于维持肺泡的正常结构和功能具有重要意义。SIRT1信号通路在细胞衰老中起着至关重要作用。本项目通过基因测序、lncRNA芯片等研究发现慢阻肺患者肺组织SAL-RNA2和SAL-RNA3表达水平显著升高，而SAL-RNA1表达水平下调；慢阻肺组肺组织较对照组SA β-gal活性显著增加、AECII凋亡水平显著升高，SIRT1及FOXO3α表达显著下降，p53及p21表达显著升高。吸烟使AECII SA β-gal活性显著增加，SIRT1和FoxO3 mRNA表达显著降低，而p53、p21 mRNA及蛋白表达显著升高；同时MMP-9 mRNA表达显著升高，TIMP-1 mRNA表达显著降低。本项目以h-SAL-RNA1过表达重组慢病毒感染AECII细胞，研究发现：重组慢病毒介导的lncRNA SAL-RNA1过表达能降低CSM诱导的AECII细胞SA-β-gal的活性，使AECII细胞SIRT1和FoxO3表达升高，而p53和p21表达降低。SRT2104使AECII细胞FoxO3、TIMP-1 mRNA表达明显升高，而p53、p21及MMP-9 mRNA表达明显降低，而SIRT1 mRNA的表达无明显变化；同时SRT2104使AECII细胞SIRT1、FoxO3、TIMP-1蛋白表达显著升高，而p53、p21 及MMP-9蛋白表达显著降低。制备C57BL/6J小鼠肺气肿模型及特异性肺Sirt1基因敲除小鼠肺气肿模型。以SRT2104治疗肺气肿C57BL/6J小鼠后，与模型组相比，SRT2104治疗组肺组织FOXO3a及 TIMP-1 mRNA表达显著升高，且p53、p21及MMP-9 mRNA表达显著降低，而Sirt1 mRNA表达无明显变化；且肺气肿小鼠肺组织Sirt1、FoxO3、TIMP-1蛋白表达显著升高，p53、p21 及MMP-9蛋白表达则显著降低。与肺Sirt1基因敲除小鼠肺气肿模型组比较，SRT2104治疗后其肺组织Sirt1、FOXO3a、p53、p21及MMP-9 mRNA与蛋白表达均无明显变化，但TIMP-1mRNA及蛋白表达显著升高。因此，SRT2104可能通过SIRT1/FoxO3a和SIRT1/p53信号通路减轻吸烟所诱导的肺气肿小鼠肺组织损伤，提示其对于慢阻肺有潜在的临床治疗价值。