细胞质靶向辐射激活Nrf2抗氧化途径的机制及作用研究

The discovering of “non-targeted effects of radiation” breaked the traditional idea that only direct hits on cell nucleus could result in genetic toxicity. Cytoplasmic radiation resulted in genetic mutation, but inflicted minimal cytotoxicity, which made it closely related to carcinogenesis. Previous studies confirmed that free radical species played pivotal roles in the process. However, the underlying mechanisms still remain largely unknown. We submit this research proposal based on our experimental results. In the current proposal, by using human lung fibroblast WI-38 and with the aid of microbeam cellular precise radiation facility, we will study the response of Nrf2 antioxidative pathway to cytoplasmic radiation and the corresponding signaling pathways. The role of mitochondria derived free radical species in cytoplasmic radiation induced Nrf2 pathway activation and DNA double strand breaks will also be explored. Furthermore, the roles of mechanisms of Nrf2 pathway in ameliorating cytoplasmic radiation induced DNA double strand breaks will be investigated. The expected results of this study will be of value for clarifying the inter-relationship between the induced biological effects induced by cytoplasmic radiation and cellular anti-oxidative responses. At the same time, the results will provide new experimental data for better understanding of the non-targeted effects of extra-nucleus targeted radiation, and for setting countermeasures against the new-born tumors during radio-therapy.

辐射非靶效应的发现打破了“对细胞核的直接作用造成辐射遗传毒性”这一传统认识。细胞质受到辐射会造成基因突变，但对细胞致死性低，导致其与肿瘤发生密切相关。已有的研究结果表明活性自由基在胞质照射生物效应过程中发挥重要作用，但其中的生物学过程和机制尚有待深入研究。在课题组前期研究基础上，本申请拟以人肺成纤维细胞WI-38为对象、Nrf2抗氧化途径为核心，利用微束细胞精确照射技术进行细胞质靶向辐射，研究Nrf2途径对胞质照射的应激响应及信号调控机理，考察胞质照射引发的线粒体自由基与Nrf2途径应激响应及细胞核DNA双链断裂损伤的关系，探讨Nrf2途径减轻胞质照射诱导细胞核DNA双链断裂损伤的机制，进而揭示胞质照射生物效应与细胞氧化应激的联系。研究结果将为进一步认识辐射非靶效应，以及在放疗过程中建立新生肿瘤预防措施提供新的实验和理论依据。

辐射非靶效应的发现打破了“对细胞核的直接作用造成细胞遗传毒性”这一传统认识。细胞质受到辐射会造成基因突变，导致其与肿瘤发生密切相关。已有的研究结果表明活性自由基在胞质照射生物效应过程中发挥重要作用，但其中的生物学过程和机制尚有待深入研究。本研究以人肺成纤维细胞WI-38为对象、NRF2抗氧化途径为核心，利用微束细胞精确照射技术进行细胞质靶向辐射，研究NRF2途径对胞质照射的应激响应及信号调控机理，考察胞质照射引发的线粒体自由基与NRF2途径应激响应及细胞核DNA双链断裂损伤的关系，探讨NRF2途径减轻胞质照射诱导细胞核DNA双链断裂损伤的机制，进而揭示胞质照射生物效应与细胞氧化应激的联系。本项目的四年研究期内，（1）发现胞质照射激活NRF2抗氧化应激途径，促进NRF2由细胞质向细胞核转运及其下游靶基因HO-1表达上调；（2）鉴定了胞质照射激活NRF2的信号途径包括两条：1）p53调控细胞线粒体片段化促进了胞质照射后NRF2由细胞质向细胞核转运积累；2）胞质照射促进了MAPK-ERK 1/2的磷酸化激活，MAPK-ERK 1/2促进了NRF2的质核转运；（3）发现胞质照射导致胞内线粒体超氧阴离子自由基水平上升。线粒体超氧阴离子自由基抑制了p53基因的表达，同时促进了线粒体片段化和NRF2向细胞核的转运和富集；此外，上升的线粒体超氧阴离子自由基促进MAPK-ERK 1/2的磷酸化激活，并导致NRF2向细胞核的转运；（4）发现胞质照射导致的NRF2氧化应激途径的激活能够促进DNA双链断裂损伤修复关键蛋白RAD51和XRCC4的细胞核累积量，与NRF2氧化应激途径减缓辐射损伤水平相关。我们的研究为辐射生物效应研究中“细胞核外靶”对辐射损伤效应调控的认识提供新的知识，相关研究结果已经在国际肿瘤学、医学等领域期刊发表标注本项目资助号的SCI论文5篇，另有1篇在审。与此同时，本研究过程中的一些新发现，如胞质照射下调p53表达的机理、胞质照射诱导线粒体转录因子A向细胞外分泌的过程和作用等，相关研究将继续开展。