癌基因诱导衰老细胞分泌的因子在体细胞重编程的机理探讨

Overexpression of defined factors Oct4, Sox2, Klf4, cMyc or Lin28 and Nanog can reprogram somatic cells into induced pluripotent stem (iPS) cells. However, almost all these factors are oncogenes, overexpression of which activates DNA damage response and induces apoptosis or senescence, posing a barrier to reprogramming. Although oncogene-induced senescence has been shown to be a roadblock to reprogramming, the role of senescent cells during reprogramming remains unknown. During early tumorigenesis, senescent cells induced by activation of oncogene have been shown to secret inflammatory factors such as IL6 and IL8 that relay and reinforce OIS. Our preliminary results revealed the elevated expression of IL6 and IL8 during reprogramming and showed that reduced IL6 expression can increase reprogramming efficiency. Therefore, the present proposal aims to systematically characterize the identity of factors secreted by senescent cells during reprogramming and examine the molecular mechanisms by which they influence reprogramming. This study will provide new understanding of the molecular mechanisms governing reprogramming and develop novel non-viral methods to improve reprogramming efficiency. Moreover, this study may shed light on developing novel strategies to delay ageing process to prolong life span.

体细胞重编程的分子调控机制仍然大部分未知.现有的研究表明:过度表达重编程因子(其中大部分是原癌基因)产生DNA损伤反应导致细胞衰老(Oncogene-Induced Senescence, OIS).抑制OIS能显著地提高重编程效率,但是衰老细胞本身对重编程的影响未知。以前在早期肿瘤发生的研究表明OIS产生的炎症因子能够加强和接力OIS.我们的前期试验表明细胞重编程过程中IL6和IL8高表达,降低IL6可以提高重编程效率。所以本研究假设重编程过程中衰老细胞所分泌的炎症因子网络能够加强和传递衰老信号从而影响重编程。阻断其信号转导机制能够提高重编程效率。该研究拟用前期成功建立的重编程模型(Stem Cells 2012)，通过分子和细胞生物学技术研究细胞衰老分泌因子影响重编程的机制。研究发现将为重编程分子机制提供新知识,为提高重编程效率提供新的手段,并将可能为延缓细胞乃至机体衰老提供新的思路。

通过本课题的实施，完成了预定目标。人体细胞重编程的分子机制大部分未知。本课题主要研究细胞衰老及衰老细胞分泌因子在人体细胞重编程中的作用机理。 首先，我们利用慢病度载体过表达OCT4, SOX2, KLF4和c-MYC重编程人成纤维细胞为iPSCs。以此为实验体系，我们通过衰老相关beta半乳糖苷酶(SA-beta-GAL)染色的方法明确了过表达重编程因子后诱导细胞衰老的发生。其次，我们发现过表达重编程因子后引起了氧化应激(oxidative stress) 导致了DNA损伤，重编程过程中添加抗氧化剂可以减少氧化应激导致的DNA损伤，保护基因组稳定性(研究结果发表Stem Cell Reports 2014, Stem Cells 2015)。最后， 我们利用FUCCI系统实现了对活的衰老细胞的分选 (研究结果发表在Scientific Reports 2016, Oncotarget 2016), 并利用qPCR检测到人体细胞重编程过程中衰老细胞分泌的因子IL6和IL8的表达上调，然后利用shRNA沉默IL6的表达后可以增加重编程效率。该研究表明过表达重编程因子引起的氧化应激导致的DNA损伤可能是引起细胞衰老，衰老细胞分泌的因子如IL6表达上调并抑制重编程发生的重要原因。该研究深入研究了氧化应激，DNA损伤，细胞衰老及衰老细胞分泌的因子在人体细胞重编程中的作用，为提高人体细胞重编程效率以及人iPSCs质量提供了新的手段。