【作者】 田一虹；

【导师】 张迎梅；

【作者基本信息】 兰州大学 ， 动物学， 2013， 硕士

【摘要】 亚精胺是广泛分布于生物体内的一种多胺类物质,在细胞增殖、机体衰老等生物学过程中发挥重要作用,其在诱导自噬方面的作用成为近年来的研究热点。研究表明,外源性亚精胺可诱导细胞自噬、抑制细胞增殖,但其是否能降低癌细胞活力及其诱导的自噬与降低癌细胞活力之间是否具有相关性的研究还未见报道。本文旨在研究亚精胺诱导癌细胞自噬及其诱导的自噬对细胞活力的影响,探究亚精胺诱导癌细胞自噬与活性氧自由基(ROS)的调控关系,探讨线粒体在亚精胺诱导的癌细胞自噬以及对细胞活力影响中的作用。本研究以人宫颈癌Hela细胞为实验材料,以亚精胺为处理药物,结合自噬抑制剂三甲基腺嘌呤进行了以下研究：用噻唑蓝比色法检测亚精胺及其诱导的自噬对细胞活力的影响；用透射电子显微镜观察亚精胺诱导细胞内形成自噬体或自噬溶酶体的情况；用蛋白质免疫印记技术检测亚精胺对细胞内自噬标志性分子LC3蛋白表达的变化；用流式细胞术检测亚精胺诱导的自噬对细胞内活性氧自由基的影响及细胞内线粒体膜电位的变化。通过研究,得到以下实验结果：不同浓度亚精胺(0、0.1、0.5、1.0mM)处理Hela细胞8h后均显著抑制了Hela细胞的活力(p<0.05),0.5mM亚精胺使细胞活力下降至对照组的65.45%;0.5mM亚精胺处理Hela细胞8h后,在透射电镜下观察到自噬体和自噬溶酶体形成,自噬标志性蛋白LC3的表达变化(LC3-II/LC3-I)为对照组的1.83倍(p<0.05)；0.5mM亚精胺与自噬抑制剂联合处理Hela细胞8h,细胞活力显著上升了9.75%(p<0.05)；0.5mM和1.0mM亚精胺处理Hela细胞8h,细胞内ROS含量分别显著下降14.18%和15.36%(p<0.05),亚精胺与自噬抑制剂联合处理细胞后,ROS水平显著升高(p<0.05),但不能完全恢复：0.5mM和1.0mM亚精胺连续处理细胞8h后,线粒体膜电位均分别显著下降至对照组的62.59%和61.90%(p<0.05)。综上所述,本研究证明了亚精胺诱导Hela细胞发生的自噬性死亡为降低细胞活力的原因之一,其诱导自噬可调控细胞内部分ROS,而亚精胺造成的线粒体膜电位急剧下降可能介导了对细胞自噬的诱导和对细胞活力的影响。更多还原

【Abstract】 Spermidine, a polyamine widely distributed in organisms, plays an important role in many biological processes such as cell proliferation, organism aging, etc.. Recent years, autophagy induced by spermidine has become a well focused study area. Previous studies have shown that exogenous spermidine can induce autophagy and inhibit cell proliferation, but few reports are concern about whether spermidine could inhibit viability of cancer cells. There are also no studies about the relationship between spermidine-induced autophagy and the reduction of cancer cell viability. The present research aimed to explore the effect of spermidine-induced autophagy on cancer cell viability, as well as the regulation between spermidine-induced autophagy and intracellular ROS. In addition, the role of mitochondria in spermidine-induced autophagy and reduction of viability in cancer cells was also investigated.This study selected Hela cells as experimental material, spermidine as a drug. Viability of Hela cells treated with spermidine or spermidine combined with autophagy inhibitor3-Methyladenine (3-MA) was measured using MTT assay. The formation of autophagysomes or autolysosomes induced by spermidine in cells was observed by transmission electron microscopy. The expression of LC3, the classical indicator of autophagy, was detected by Western Blot. Intracellular ROS content and mitochondrial membrane potential was determined by flow cytometry.Through the above research, we draw the following experimental results. Exposure to0.1mM,0.5mM and1.0mM spermidine for8h significantly inhibited Hela cell vitality (p<0.05).0.5mM spermidine decreased cell viability to65.45%compared with control group. Autophagosomes and autolysosomes induced by0.5mM spermidine for8h can be observed through transmission electron microscope. In addition, the ratio of LC3-II/LC3-I (autophagy marker protein) in0.5mM spermidine group was1.83fold higher than that of control (p<0.05). Cell viability was increased by9.75%(p<0.05) when Hela cells were exposed to0.5mM spermidine combined with5.0mM3-MA for8h. Moreover, intracellular ROS level was significantly reduced by14.18%(p<0.05) and the mitochondrial membrane potential was respectively declined to62.59%and61.90%when Hela cells were treated with0.5mM and1.0mM spermidine for8h. However, once autophagy was inhibited, the content of ROS was prominently rescued by about10%(p<0.05). In summary, our study proved spermidine-induced autophagic death was one of the reasons to reduce Hela cell viability. Autophagy induced by spermindine could regulate ROS partly. The sharp decline of mitochondrial membrane potential caused by spermidine might mediate the induction of autophagy and reduction of cell viability.更多还原