【作者】 周莹；

【导师】 刘耕；

【作者基本信息】 南京大学 ， 遗传学， 2016， 硕士

【摘要】 细胞死亡和存活是细胞命运的重要组成和话题,被很多因素影响。活性氧(ROS, reactive oxygen species)是细胞线粒体氧化磷酸化生产能量过程中生成的有毒副产品,能破坏线粒体和细胞DNA、蛋白质、脂类和其他分子导致氧化应激。氧化应激的增加足以造成细胞衰老。在体外培养的正常人类成纤维细胞和小鼠胚胎成纤维细胞(MEF, Mouse Embryonic Fibroblast)中可以观察到衰老的发生,这些细胞只有有限的细胞复制能力,并最终发生细胞阻滞停止生长。原癌基因C-myc过表达,抑癌基因p53缺失以及一些糖酵解代谢基因的过表达能够实现对MEF细胞的重编程,抑制ROS增多和细胞毒性引起的细胞衰老,改变MEF细胞的代谢和命运使得细胞发生永生化。转录因子NRF2的主要功能是通过诱导一些下游基因的转录来调控一系列重要的抗氧化基因的表达,从而保护细胞免受外源性和内源性有害异物和氧化应激的影响。由此,我们想研究NRF2在氧化应激所诱导的MEF细胞衰老中作用,以及在怎样的条件下如何调节氧化应激平衡来调控不同的细胞命运。我们利用慢病毒在MEF细胞中过表达抗氧化转录因子NRF2的突变体,持续激活NRF2的表达,检测细胞生长水平和发生永生化的情况。我们的实验结果表明,在早期传代的MEF(P6代之前)中过表达抗氧化基因NRF2不利于细胞的增殖生长。细胞内ROS水平显著降低,细胞增殖减少,衰老增加。但随着代数的增加,体外培养细胞的内在和外在氧化应激和有毒物质的增多,一部分永生化的MEF细胞内ROS逐渐累积达到较高水平,维持ROS不会过高引起细胞损伤,但却可以促进细胞生存和增殖。因此,在不同时期的MEF细胞中激活的NRF2,通过调控细胞氧化应激水平对细胞命运具有双重作用。另一方面,过表达了NRF2的早期传代MEF中,线粒体膜电位和ATP产量降低,提示NRF2可以参与调控细胞代谢和线粒体功能。此外,另一个重要的抗氧化基因FOXO3的表达可以一定程度上延长细胞寿命,而与NRF2共同过表达时可以挽救NRF2对细胞生长的影响。这些线索有助于我们进一步揭示NRF2对细胞影响的更多的内容。更多还原

【Abstract】 Cell death and survivor are important parts of Cell fate, which are affected by many factors. ROS generated as a toxic by-product of oxidative energy production by OXPHO; which damage the mitochondrial and cellular DNA, proteins, lipids, and other molecules causing oxidative stress. An increase in oxidative stress has independently been implicated in cellular senescence. Senescence can be observed in normal human fibroblasts and Mouse Embryonic Fibroblast (MEF) in culture, which have a finite replicative life span and then become permanently arrested. The expression of proto-oncogene c-myc, loss or mutations of tumor suppressor gene p53, as well as some overexpression of glycolysis metabolism genes can achieve the reprogramming of cells, shift metabolism and destiny of MEF, inhibit the increase in ROS and cell toxicity caused by aging, so as to make the cells occur immortality.The main function of the transcription factor NRF2 is to activate the cellular antioxidant response by inducing the transcription of several genes to protect cells from the effects of exogenous and endogenous insults such as xenobiotics and oxidative stress. NRF2 also affects multiple aspects of intermediary metabolism and mitochondrial function, and so influences the synthesis of carbohydrates, nucleic acids, lipids, and amino acids. In view of the implication of oxidative stress in MEF senescence and the capacity of NRF2 to reduce oxidative stress levels, affect metabolism, protect cells from various stresses, we studied the involvement of NRF2 in oxidative stress-induced senescence and how conditions of NRF2 expression may modulate oxidative stress balance to achieve different cell fate outcomes.We over express mutant oxidation resistance transcription factor NRF2 by lentivirus in MEF cells, activate expression of NRF2 downstream genes, and then detect the cell growth and cell immortalized situation. Our experimental results show that, the overexpression of NRF2 is not conducive to the proliferation of cell growth in the early stage of the MEF. We find that the cells have significantly suppressed intracellular ROS level, decreased cell proliferation and increased senescence. However, as the oxidative stress and toxic substances of inner and outer cell increased within the cultured cells in vitro, strengthening NRF2 regulating the antioxidant capacity is beneficial to the growth and survival of cell. Therefore, the cells have different fates by expressing NRF2 to inhibit oxidative stress level in the different periods of MEFs.On the other hand, the mitochondrial membrane potential and ATP production also decrease in the early stage of the MEF with NRF2 overexpression, which reflect that the NRF2 take part in the regulation of cell metabolism and mitochondrial function. Surprisingly, the expression of another important oxidation resistance gene FOXO3 can extend the limited life span of cells to some extent, and rescue NRF2 effects on cells, which suggest the effects of the two factors on cell fate are different and may have some relationships. These clues will help us to further reveal more content about the NRF2 effects on cells.更多还原