诱导多功能干细胞（induced pluripotent stem cells，iPSCs）具有来源简易和分化潜能高等特点，已被广泛用于基础和临床相关研究。但iPSCs的四倍体补偿实验成功率较低，说明其质量不高，因此iPSCs距离临床应用仍存在障碍。基于iPSCs连续诱导重编程六次而获得的六代完全由iPSCs发育而来成活的“all-iPSCs”小鼠研究显示，“all-iPSCs”小鼠活力可能由于突变逐步增加而逐渐减弱。利用该系统，本课题组初步数据显示，不同DNA损伤修复通路效率减弱，相关机制研究显示SIRT2、SIRT6可参与调控这些通路。因此，申请人希望阐述三个关键科学问题，1. iPSCs质量下降是否由DNA修复效率下调使得基因组不稳定性增高导致？2. 哪条DNA损伤修复通路更重要？分子机制是什么？3. 激活SIRT2是否可以提高iPSCs质量？

Induced pluripotent stem cells (iPSCs) have been widely utilized in basic and clinical research due to the features of easy-to-obtain and high pluripotency potentials. However, the low successful rate of tetraploid complementation experiments using iPSCs indicates that the quality of iPSCs is incomparable to that of embryonic stem cells. A recently developed model of sequentially reprogrammed induced pluripotent stem cells demonstrated that the number of mutations accumulate with increasing generation of all-iPSCs mice while the viability of the all-iPSCs mice decreases with it. Using this model, our preliminary data indicates that the efficiency of some DNA repair pathways declines with the increasing generations. Mechanistic studies reveal that SIRT2 and SIRT6 participate in the regulation of DNA repair pathways. We therefore propose the following three aims in this study. Aim 1: To examine whether the reduced repair efficiency leads to the increased genomic instability, eventually reducing the iPSCs quality. Aim 2: To examine which repair pathways are responsible for the rise of the genomic instability and the underling regulatory mechanisms. 3. To examine whether activating SIRT2 can improve the quality of iPSCs.

六代体系中，小鼠的生存能力随着重编程代数的上升而下降，这与重编程过程中不断积累的点突变息息相关。本课题研究发现发现连续重编程的iPS细胞的基因组稳定性逐步下调，其中碱基切除修复效率随代数增加显著下调而DNA双链断裂损伤修复能力没有发生变化。通过蛋白水平的分析，我们发现APE1以及衰老相关蛋白如Sirt2等重要碱基切除修复蛋白的表达随重编程次数逐步下调。同时，机制性的研究表明Sirt2可以通过其去乙酰化酶活性调控碱基切除修复，且 Sirt2与碱基切除修复通路中重要的Ogg1蛋白相互作用并去乙酰化该蛋白调控其进核，此外Sirt2结合至Ogg1的启动子促进Ogg1的表达，这些调控均促进碱基切除修复效率。综上，本课题揭示了连续诱导重编程过程中不断积累的点突变与BER效率的降低密切相关，而Sirt2可能起着重要的调控作用。相关研究提示我们可发展激活Sirt2的小分子化合物进而提高碱基切除修复效率维持基因组稳定性，最终促进iPS细胞的质量的提升。