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首页> 外文学位 >Antioxidant defense and redox responses to telomere homolog oligonucleotides in human dermal fibroblasts: A model for investigating redox signaling responses to DNA damage.
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Antioxidant defense and redox responses to telomere homolog oligonucleotides in human dermal fibroblasts: A model for investigating redox signaling responses to DNA damage.

机译：对人类皮肤成纤维细胞中端粒同源寡核苷酸的抗氧化防御和氧化还原反应：研究对DNA损伤的氧化还原信号反应的模型。

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It has been demonstrated that oligonucleotides homologous to the 3 ' telomere repeat sequence TTAGGG (T-oligos) stimulate DNA damage responses that are also induced by disruption of the telomere loop structure. Adaptive defense against oxidative stress and UV or ionizing radiation has been reported, but adaptive antioxidant defense as a response to mimicking telomere loop exposure has not been described. The T-oligos pTT and pGTTAGGGTTAG were added to human dermal fibroblast cultures to investigate whether mimicking telomere loop disruption stimulates antioxidant defense. pTT stimulated mitochondrial superoxide dismutase protein levels within 72 hours. Cell yields were higher after H2O2 exposure in fibroblasts pretreated with pTT for 72 hours compared to diluent pretreated cells. Intracellular reactive oxygen species (ROS) levels, measured by flow cytometry and the dichlorofluorescein diacetate probe, increased during T-oligo treatment as compared to diluent and oligonucleotide controls. The time course and degree of ROS stimulation corresponded to the time course for activation and/or induction of p53 and p21/Cip1/Waf1. The NADPH oxidase inhibitor diphenyliodonium chloride abrogated this increase and fibroblasts retrovirally transduced to produce dominant negative p53 failed to display increased ROS, implicating that the T-oligos induced ROS through p53-responsive NADPH oxidases. A horseradish peroxidase assay for extracellular H2O2 showed no H2O 2 release with pTT treatment. To determine whether there was induction of senescence, an endpoint response to increased ROS and prolonged T-oligo treatment in fibroblasts, the senescence-associated beta-galactosidase assay was conducted in parallel with the DCF assay. Only the 11mer T-oligo treatment modestly increased the number of beta-galactosidase positive cells by 72 hours (30% of cells). This is the first report suggesting that antioxidant defense and ROS signaling are part of the broad adaptive response in mammalian cells presumably initiated by telomere loop disruption and mimicked by T-oligos. T-oligo treatment thus offers a new model for studies of ROS signaling in human dermal fibroblasts, allowing exploration of the relationships between DNA damage, ROS, oxidative stress, and the evolution of cellular defense mechanisms.
机译：已经证明，与3'端粒重复序列TTAGGG（T-寡核苷酸）同源的寡核苷酸刺激DNA损伤反应，其也由端粒环结构的破坏诱导。已经报道了针对氧化应激和紫外线或电离辐射的自适应防御，但是尚未描述作为对模仿端粒环暴露的响应的自适应抗氧化剂防御。将T-寡核苷酸pTT和pGTTAGGGTTAG添加到人皮肤成纤维细胞培养物中，以研究模拟端粒环破坏是否刺激抗氧化防御。 pTT在72小时内刺激了线粒体超氧化物歧化酶蛋白水平。与稀释剂预处理的细胞相比，经pTT预处理72小时的成纤维细胞中的H2O2暴露后，细胞产量更高。与稀释剂和寡核苷酸对照相比，在T-寡核苷酸处理期间，通过流式细胞仪和二氯荧光素二乙酸酯探针测量的细胞内活性氧（ROS）水平增加。 ROS刺激的时间过程和程度对应于激活和/或诱导p53和p21 / Cip1 / Waf1的时间过程。 NADPH氧化酶抑制剂二苯基氯化碘氯化物消除了这种增加，逆转录病毒转导产生显性负性p53的成纤维细胞未能显示出增加的ROS，暗示T-寡核苷酸通过p53反应性NADPH氧化酶诱导了ROS。辣根过氧化物酶测定细胞外H2O2的结果表明，pTT处理不会释放H2O2。为了确定是否存在衰老诱导，对成纤维细胞中ROS升高和T-寡核苷酸治疗延长的终点反应，将衰老相关的β-半乳糖苷酶检测与DCF检测并行进行。仅11mer T-寡核苷酸处理可适度增加β-半乳糖苷酶阳性细胞的数量72小时（<30％的细胞）。这是第一个报告，表明抗氧化剂防御和ROS信号是哺乳动物细胞中广泛的适应性反应的一部分，该反应可能是端粒环破坏引起的，并被T-寡核苷酸所模仿。因此，T-寡核苷酸治疗为研究人类皮肤成纤维细胞中的ROS信号提供了新的模型，从而可以探索DNA损伤，ROS，氧化应激和细胞防御机制之间的关系。

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