阿维菌素的细胞毒性及分子机理研究

发布时间：2018-03-24 02:34

本文选题：阿维菌素　切入点：细胞毒性　出处：《华东理工大学》2017年博士论文

【摘要】：阿维菌素作为生物农药家族最主要的杀虫剂成员之一,广泛应用于农林业生产领域,并在提高农作物产量等方面起到了极大的作用。随着阿维菌素的大量使用,人们对其毒理学效应的关注度也越来越高。作为农用杀虫剂,在杀虫机理方面的研究只明确认为是一种神经毒剂,但确认其并非具有唯一的作用靶标。同时,国内外越来越多的报道说明了阿维菌素在环境暴露中引起的毒性作用,其中大部分研究聚焦于阿维菌素对施用环境中非靶标生物的毒理学影响,而鲜见对人体细胞的毒性研究。本论文聚焦于阿维菌素的细胞毒性及分子机理,针对以Sf9细胞为代表的昆虫体细胞和以HeLa细胞、HepG2细胞为代表的人体细胞开展了系统性的体外细胞毒性实验进行研究。利用MTT实验和细胞克隆形成实验研究发现,阿维菌素具有降低细胞存活率、抑制细胞增殖的毒性影响,且对三种代表细胞无显著的选择性差异。利用单细胞凝胶电泳检测DNA损伤和用荧光免疫印迹法对组蛋白H2AX磷酸化的研究中发现,阿维菌素可以诱导细胞发生DNA损伤,并且具体表现为DNA双链断裂的严重损伤作用。针对线粒体及活性氧自由基的研究中发现,阿维菌素可以诱导线粒体的肿胀及细胞内活性氧自由基的产生和积累。通过增加抗氧化剂的方法研究发现,阿维菌素造成的活性氧自由基的产生与积累是引起DNA链发生损伤的主要诱因。在对阿维菌素引起的DNA损伤中研究发现,DNA链上受损碱基与嘌呤或甲酰胺基嘧啶相关;阿维菌素致使DNA受损的过程还引起了8-羟基脱氧鸟苷(8-oxodG)的产生,而该物质会使DNA链发生空间结构的改变,这种改变具有引起突变和诱发癌变的潜在毒性。对DNA损伤监控和修复关键蛋白PARP的试验检测中发现,阿维菌素引起了 PARP的被切割,其中24kD多肽和DNA的持续性结合将会导致DNA不能被修复,引起细胞死亡。而PARP的切割主要来源于细胞凋亡过程中caspase3的活化,这预示着阿维菌素引起DNA损伤后,很可能通过凋亡机制的启动,完成对细胞增殖的抑制或者引起细胞死亡。通过Hoechst染色实验、AO/EB染色实验、Annexin V-FITC/PI双染和流式细胞术研究证实,阿维菌素具有诱导细胞凋亡的能力。通过对线粒体膜电位、细胞色素C、caspase9、caspase3、Bax/Bcl-2的研究发现,阿维菌素对细胞凋亡的诱导与线粒体信号转导途径相关;而利用caspase特异性抑制剂抑制caspase3活性后,发现细胞凋亡比率下降,这也说明阿维菌素诱导细胞凋亡具有caspase依赖特性。利用透射电子显微镜检测及MDC染色发现,阿维菌素诱导下的细胞内出现了典型的自噬特征,而在进一步对自噬发生标志蛋白LC3Ⅰ/Ⅱ、Beclin1、p62的试验检测中确认了自噬的发生。通过对细胞ATP含量的检测及对自噬信号转导通路相关蛋白mTOR、AMPK、p70s6k的磷酸化表达研究中发现,阿维菌素可能通过对线粒体的损伤作用,降低了细胞ATP含量从而激活了 AMPK/mTOR介导的自噬信号通路,进而通过影响下游的p70s6k的磷酸化水平对细胞的生长、增殖和存活产生影响。针对昆虫及人体不同细胞系,探索阿维菌素的细胞毒性及分子机理,有助于揭示阿维菌素的潜在杀虫机理,为新农药创制奠定科学基础;更有助于明确阿维菌素对人体健康的潜在威胁,为日益凸显的农药安全问题提供深层次有效评价方法。
[Abstract]:As one of the main members of abamectin pesticide bio pesticide family, widely used in agriculture and forestry production areas, and in increasing agricultural productivity has played a great role. With the extensive use of abamectin, the toxicological effects of attention are also increasing. As an agricultural insecticide, on the insecticidal mechanism just think clearly is a nerve agent, but has not only confirmed the target. At the same time, more and more domestic and foreign reports indicate that the toxicity of Abamectin in environment caused by exposure, most of the research focus in the toxicological effects of Abamectin on Application in environment of non target organisms, and the toxicity of rare human cells. This paper focus on the cytotoxicity of avermectin and molecular mechanism for insect cell line by Sf9 cells as the representative and the HeLa cell, He PG2 cells represented human cells carried out in vitro cytotoxicity test system was studied. The formation of experimental study found that using MTT assay and cell cloning, avermectin can reduce the cell survival rate, toxicity effect of inhibiting cell proliferation, no significant differences in selectivity and three representative cells. Using single cell gel electrophoresis to detect DNA damage research and discovery on histone H2AX phosphorylation by fluorescent immunoblotting, abamectin can induce DNA damage, and the specific performance of serious damage effect of DNA double strand breaks. According to the research of mitochondria and active oxygen free radicals that ROS can induce mitochondrial swelling of Abamectin and cell in the production and accumulation. By increasing the research methods of antioxidants, production and accumulation of active oxygen free radicals caused by the avermectin is caused by DN A chain main inducement of injury. The study found that the DNA damage caused by abamectin, DNA chain damaged bases and purine or pyrimidine carboxamides; avermectin causes DNA to the process of damage caused 8- hydroxydeoxyguanosine (8-oxodG) production, and the material will make the DNA chain space structure change, this change has caused the potential toxicity and mutation induced cancer. Found the test on DNA damage monitoring and repair key protein PARP, abamectin caused a PARP cut, which will cause the DNA cannot be repaired with persistent 24kD polypeptide and DNA, causing cell death. Activation of PARP cutting from Caspase3 in the process of cell apoptosis, which indicates that the DNA damage caused by Abamectin, probably through the mechanism of apoptosis initiation, complete inhibition of cell proliferation or induce cell death. Hoechst staining, AO/EB staining experiment, Annexin V-FITC/PI double staining and flow cytometry studies confirmed that abamectin has the ability to induce apoptosis. The potential of mitochondrial membrane, cytochrome C, caspase9, Caspase3, Bax/Bcl-2 study found that avermectin of cell apoptosis is associated with mitochondrial signal transduction pathway and inhibition of Caspase3; activity with specific inhibitors of caspase, decrease the percentage of apoptotic cells, which also shows that the avermectin induced apoptosis with caspase and MDC. The dependence on measured using transmission electron microscopy staining showed that avermectin cells induced by autophagy in typical characteristics, and further to the autophagy marker protein LC3 1 / 2, Beclin1 the test of p62, confirmed the autophagy. Through the detection of cell ATP content and signal transduction pathway related to autophagy. Protein mTOR, AMPK, found on the expression of phosphorylated P70S6K in avermectin may effect on mitochondria by damage, reduce the content of ATP cells to activate autophagy signaling pathway mediated by AMPK/mTOR, and then through the influence of lower the level of phosphorylation of P70S6K on cell growth, proliferation and survival impact. For insects and human body different cell lines and explore the molecular mechanism of cell toxicity and abamectin, helps to reveal the potential Insecticidal Mechanism of avermectin, lay a scientific foundation for the creation of new pesticides; helps clear avermectin a potential threat to human health, provide deep effective evaluation method for pesticide safety problems have become increasingly prominent.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ450.26

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